Combination therapy for treating or preventing cancer

ABSTRACT

The invention provides a combination therapy comprising a bacterial strain for treating or preventing cancer.

CROSS REFERENCE

This application is a continuation of U.S. Non-Provisional applicationSer. No. 16/931,104, filed on Jul. 16, 2020, which is a continuation ofInternational Application No. PCT/GB2019/050143, filed on Jan. 18, 2019,which claims priority to GB Application No. 1800927.4, filed on Jan. 19,2018, GB Application No. 1801502.4, filed on Jan. 30, 2018, GBApplication No. 1805941.0, filed on Apr. 10, 2018, GB Application No.1806572.2, filed on Apr. 23, 2018, and GB Application No. 1808631.4,filed on May 25, 2018, each of which is herein incorporated by referencein its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in Extendible Markup Language (XML) format andis hereby incorporated by reference in its entirety. Said XML copy,created on Sep. 27, 2022, is named 56708-733302.xml and is 5,743 bytesin size.

TECHNICAL FIELD

This invention is in the field of a combination therapy for treating orpreventing cancer: a combination of a composition comprising a bacterialstrain and an inhibitor selected from the group consisting of Nivolumab,BGB-A137, cemiplimab, PDR001, camrelizumab, BCD-100, IBI308, AGEN2034,INCSHR1210, MEDI0680, JS001/PD1, CC-90006, BI 754091, JNJ-63723283,PF-06801591, GLS-010, AB122, Sym021, MGA012, LZMO09, genolimzumab,AK105, AB122, PF-06801591, PF-06688992 and TSR-042, for treating orpreventing cancer.

BACKGROUND TO THE INVENTION

The human intestine is thought to be sterile in utero, but it is exposedto a large variety of maternal and environmental microbes immediatelyafter birth. Thereafter, a dynamic period of microbial colonization andsuccession occurs, which is influenced by factors such as delivery mode,environment, diet and host genotype, all of which impact upon thecomposition of the gut microbiota, particularly during early life.Subsequently, the microbiota stabilizes and becomes adult-like [1]. Thehuman gut microbiota contains more than 500-1000 different phylotypesbelonging essentially to two major bacterial divisions, theBacteroidetes and the Firmicutes [2]. The successful symbioticrelationships arising from bacterial colonization of the human gut haveyielded a wide variety of metabolic, structural, protective and otherbeneficial functions. The enhanced metabolic activities of the colonizedgut ensure that otherwise indigestible dietary components are degradedwith release of by-products providing an important nutrient source forthe host. Similarly, the immunological importance of the gut microbiotais well-recognized and is exemplified in germfree animals which have animpaired immune system that is functionally reconstituted following theintroduction of commensal bacteria [3-5].

Dramatic changes in microbiota composition have been documented ingastrointestinal disorders such as inflammatory bowel disease (IBD). Forexample, the levels of Clostridium cluster XIVa bacteria are reduced inIBD patients whilst numbers of E. coli are increased, suggesting a shiftin the balance of symbionts and pathobionts within the gut [6-9].Interestingly, this microbial dysbiosis is also associated withimbalances in T effector cell populations.

In recognition of the potential positive effect that certain bacterialstrains may have on the animal gut, various strains have been proposedfor use in the treatment of various diseases (see, for example,[10-13]). Also, certain strains, including mostly Lactobacillus andBifidobacterium strains, have been proposed for use in treating variousinflammatory and autoimmune diseases that are not directly linked to theintestines (see [14] and [15] for reviews). However, the relationshipbetween different diseases and different bacterial strains, and theprecise effects of particular bacterial strains on the gut and at asystemic level and on any particular types of diseases, are poorlycharacterised. For example, certain Enterococcus species have beenimplicated in causing cancer [16]. In contrast, bacterial strains of thespecies Enterococcus gallinarum have also been disclosed for use intreating and preventing cancer [54].

Due to the diverse nature of cancer, various treatment modalities arebeing developed in order to treat different patient groups. Onetreatment modality that has proved effective is the use of ImmuneCheckpoint Inhibitors (ICIs). ICIs are compounds that inhibit a cancercell's ability to prevent the host's immune cells from attacking cancercells. ICIs may be, for instance, therapeutic antibodies that have beendeveloped against the interaction between the transmembrane receptorprogrammed cell death 1 protein (referred to as PDCD1, PD-1, PD1, orCD279) and its ligand, PD-1 ligand 1 (referred to as PD-L1, PDL1 orCD274).

Although treatment of cancer patients with an ICI, when effective, canresult in long lasting and significant clinical effects, there is stilla significant percentage of patients that are non-responsive or onlypartially responsive to ICI treatment. There is therefore a requirementin the art for new and improved treatment modalities to prevent andtreat cancer, and in particular treatment modalities which may improvethe effect of treatment with particular inhibitors.

SUMMARY OF THE INVENTION

The present invention relates to novel combination therapies fortreating and preventing cancer. In particular, the present inventionrelates to improved therapies in which sequential and/or partiallyparallel administration of a bacterial strain of the speciesEnterococcus gallinarum and an inhibitor selected from the groupconsisting of Nivolumab, BGB-A137, cemiplimab, PDR001, camrelizumab,BCD-100, IBI308, AGEN2034, INCSHR1210, MEDI0680, JS001/PD1, CC-90006, BI754091, JNJ-63723283, PF-06801591, GLS-010, AB122, Sym021, MGA012,LZMO09, genolimzumab, AK105, AB122, PF-06801591, PF-06688992 and TSR-042results in a more effective treatment of cancer than treatment with thebacterial strain or the inhibitor alone.

Compositions comprising a bacterial strain of the species Enterococcusgallinarum are effective in therapy in general, and in treating orpreventing cancer in particular, as presented herein below and in [54].The present invention is further based in part on the unexpected effectachieved upon administration of both a particular inhibitor and acomposition comprising a bacterial strain of the species Enterococcusgallinarum. As used herein, the terms “the combination of theinvention”, “the therapeutic combination of the invention” and “thetherapeutic combination” may be used interchangeably and refer to atherapeutic combination of: (a) a composition comprising a bacterialstrain of the species Enterococcus gallinarum; and (b) an inhibitorselected from the group consisting of Nivolumab, BGB-A137, cemiplimab,PDR001, camrelizumab, BCD-100, IBI308, AGEN2034, INCSHR1210, MEDI0680,JS001/PD1, CC-90006, BI 754091, JNJ-63723283, PF-06801591, GLS-010,AB122, Sym021, MGA012, LZMO09, genolimzumab, AK105, AB122, PF-06801591,PF-06688992 and TSR-042. It is to be understood that the term“combination” in the context of the therapeutic combination does notrefer to components (a) and (b) of the combination necessarily being inthe same composition and/or administered at the same time. According topreferred embodiments, (a) and (b) of the therapeutic combination are inseparate compositions. According to some embodiments, provided herein isthe combination of the invention for use in a method of treating orpreventing cancer in a subject. According to some embodiments, providedherein is a method for treating or preventing cancer in a subject,comprising administering the therapeutic combination of the invention tothe subject.

According to some embodiments, administration of the bacterialcomposition in the context of the therapeutic combination enablestreatment of cancer patients who were non-responsive or who showedinsufficient response to treatment with an immune checkpoint inhibitorthat was administered without the bacterial composition. According tosome embodiments, the patients who are non-responsive or partialresponders to ICI therapy may be ICI naïve (i.e. they have notpreviously received ICI therapy) or they may have become non-respondersor partial responders following previously successful administration ofICIs.

Without wishing to be bound by theory or mechanism, this effect might bethrough modulation of mediators that improve the efficiency ofinhibitors of the invention, such as through an increase intumour-infiltrating CD8⁺ T-cells or an increase in the ratio oftumour-infiltrating CD8⁺ T-cells to FoxP3+ cells.

According to one aspect, provided herein is a therapeutic combinationfor use in a method of treating or preventing cancer in a subject,wherein said therapeutic combination comprises:

-   -   (a) a composition comprising a bacterial strain of the species        Enterococcus gallinarum; and    -   (b) an inhibitor selected from the group consisting of        Nivolumab, BGB-A137, cemiplimab, PDR001, camrelizumab, BCD-100,        IBI308, AGEN2034, INCSHR1210, MEDI0680, JS001/PD1, CC-90006, BI        754091, JNJ-63723283, PF-06801591, GLS-010, AB122, Sym021,        MGA012, LZMO09, genolimzumab, AK105, AB122, PF-06801591,        PF-06688992 and TSR-042.

According to some embodiments, provided herein is a compositioncomprising a bacterial strain of the species Enterococcus gallinarum foruse in a method of treating or preventing cancer in a subject, whereinsaid composition is used in combination with an inhibitor of theinvention.

According to some embodiments, provided herein is a first compositioncomprising a bacterial strain of the species Enterococcus gallinarum foruse in combination with a second composition comprising an inhibitor ofthe invention, for use in a method of treating or preventing cancer in asubject, optionally wherein said first composition is administered priorto first administration of said second composition and/or in parallel tothe administration of the second composition, optionally wherein thesubject was non-responsive to a prior treatment using an immunecheckpoint inhibitor alone.

According to another aspect, provided herein is a method of treating orpreventing cancer in a subject in need thereof (referred to herein alsoas “the method of the invention”), the method comprising: (a)administering to the subject a composition comprising a bacterial strainof the species Enterococcus gallinarum; and (b) administering to thesubject an inhibitor selected from the group consisting of Nivolumab,BGB-A137, cemiplimab, PDR001, camrelizumab, BCD-100, IBI308, AGEN2034,INCSHR1210, MEDI0680, JS001/PD1, CC-90006, BI 754091, JNJ-63723283,PF-06801591, GLS-010, AB122, Sym021, MGA012, LZMO09, genolimzumab,AK105, AB122, PF-06801591, PF-06688992 and TSR-042.

According to another aspect, provided herein is a kit comprising: (a) acomposition comprising a bacterial strain of the species Enterococcusgallinarum; and (b) a composition comprising an inhibitor selected fromthe group consisting of Nivolumab, BGB-A137, cemiplimab, PDR001,camrelizumab, BCD-100, IBI308, AGEN2034, INCSHR1210, MEDI0680,JS001/PD1, CC-90006, BI 754091, JNJ-63723283, PF-06801591, GLS-010,AB122, Sym021, MGA012, LZMO09, genolimzumab, AK105, AB122, PF-06801591,PF-06688992 and TSR-042.

According to some embodiments, cancer is selected from the groupconsisting of: breast cancer, lung cancer, colon cancer, kidney cancer,liver cancer, lymphoma (such as non-Hodgkin's lymphoma), hepatoma andneuroendocrine cancer. According to some embodiments, the therapeuticcombination is for use in a method of treating or preventing lungcancer, breast cancer, kidney cancer, liver cancer, lymphoma, hepatoma,neuroendocrine cancer or colon cancer. According to some embodiments,cancer is selected from the group consisting of: melanoma, non-smallcell lung carcinoma, bladder cancer and head-and-neck cancer. In certainembodiments, the therapeutic combination or the method of the inventionis for use in reducing tumour size or preventing tumour growth in thetreatment of cancer. According to some embodiments, the therapeuticcombination or the method of the invention is for use in at least one ofreducing tumour size, reducing tumour growth, preventing metastasis orpreventing angiogenesis.

According to some embodiments, the terms “the composition”, “thebacterial composition” and “the composition of the invention” may beused interchangeably and refer to the composition included in thetherapeutic combination of the invention, which comprises a bacterialstrain of the species Enterococcus gallinarum. According to someembodiments, the composition comprising a bacterial strain of thespecies Enterococcus gallinarum does not contain bacteria from any otherspecies or comprises only de minimis or biologically irrelevant amountsof bacteria from another species. According to some embodiments, closelyrelated strains of Enterococcus gallinarum may also be used as part ofthe therapeutic combination, such as bacterial strains that have a 16srRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%identical to the 16s rRNA sequence of a bacterial strain of Enterococcusgallinarum. Preferably, the bacterial strain has a 16s rRNA sequencethat is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical toSEQ ID NO:1 or 2. Preferably, the sequence identity is to SEQ ID NO:2.Preferably, the bacterial strain for use in the therapeutic combinationof the invention has the 16s rRNA sequence represented by SEQ ID NO:2.

Accordingly, the therapeutic combination of the invention may comprise acomposition comprising a bacterial strain that has a 16s rRNA sequencethat is at least 95% identical to the 16s rRNA sequence of a bacterialstrain of Enterococcus gallinarum, optionally to SEQ ID NO: 2, for usein a method of treating or preventing cancer. Enterococcus gallinarum Insome embodiments, the bacterial strain in the composition is not ofEnterococcus gallinarum, but is a closely related strain.

In certain embodiments, the composition of the invention is for oraladministration. Oral administration of the strains of the invention canbe effective for treating cancer, in particular when administered aspart of the therapeutic combination of the invention. Also, oraladministration is convenient for patients and practitioners and allowsdelivery to and/or partial or total colonisation of the intestine.According to some embodiments, the inhibitor used as part of thetherapeutic combination of the invention is administered intravenously.According to some embodiments, each of the bacterial composition and theinhibitor of the therapeutic combination are present in a separatecomposition, each possibly comprising a carrier and/or an excipientsuitable for its mode of administration. In certain embodiments, thecomposition of the invention comprises one or more pharmaceuticallyacceptable excipients or carriers. In certain embodiments, the inhibitorof the invention is in a composition comprising one or morepharmaceutically acceptable excipients or carriers.

In certain embodiments, the bacterial composition of the inventioncomprises a bacterial strain that has been lyophilised. Lyophilisationis an effective and convenient technique for preparing stablecompositions that allow delivery of bacteria. According to someembodiments, the bacterial strain in the composition is capable ofpartially or totally colonising the intestine.

In certain embodiments, the bacterial composition is comprised in a foodproduct. In certain embodiments, the bacterial composition is comprisedin a vaccine.

According to some embodiments, the bacterial composition comprises asingle strain of Enterococcus gallinarum. According to some embodiments,the bacterial composition comprises the Enterococcus gallinarumbacterial strain as part of a microbial consortium. Preferably, thebacterial composition comprises the Enterococcus gallinarum straindeposited under accession number NCIMB 42488.

According to some embodiments of the method of the invention, thebacterial composition is administered to the subject prior to a firstadministration of the inhibitor of the invention to the subject.

According to some embodiments of the method of the invention, thebacterial composition is administered to the subject for at least one,two, three or four weeks prior to first administration of the inhibitorof the invention. It is to be understood that in the context of themethod of the invention, the first administration of the inhibitor ofthe invention refers to a first administration as part of thetherapeutic combination of the invention. Prior to administration of thetherapeutic combination of the invention the subject might have beenadministered with an inhibitor without the bacterial composition of theinvention being administered during/before administration of theinhibitor. According to some embodiments, at least one, two, three orfour weeks passed between administration of the therapeutic combinationof the invention and prior administration of an inhibitor alone or thebacterial composition alone.

According to some embodiments of the method of the invention, thebacterial composition is administered to the subject at least partiallyin parallel to administration of the inhibitor of the invention to thesubject. In the context of administration times of the bacterialcomposition and the inhibitor of the invention, administration at leastpartially in parallel refers to administrations which may overlapcompletely (for example, administration of both components over a courseof 12 months) or partially (for example, administration of one componentover a course of 12 months and administration of the second componentover a course of 8 months, which may overlap completely or partiallywith the 12 month period). It is to be understood that paralleladministration of both components does not mean that both components arenecessarily administered using the same dosage regime. According to someembodiments of the method of the invention, the bacterial composition isadministered to the subject prior to first administration of theinhibitor of the invention and/or at least partially in parallel toadministration of the inhibitor of the invention to said subject.According to certain embodiments, the bacterial composition isadministered to the subject for at least one, two, three or four weeksprior to first administration of the inhibitor of the invention,followed by administration of the bacterial composition and theinhibitor of the invention at least partially in parallel for at leasttwo, four or six weeks.

According to some embodiments, the bacterial strain of the speciesEnterococcus gallinarum and the inhibitor of the invention are inseparate compositions, preferably wherein the bacterial composition isformulated for oral administration whereas the inhibitor of theinvention is in a formulation formulated for intravenous administration.

According to some embodiments, the therapeutic combination of theinvention is for treating or preventing cancer in a subject who wasnon-responsive to a prior treatment using an immune checkpoint inhibitoralone. As used herein, a subject who is non-responsive to treatment withan immune checkpoint inhibitor relates to a subject who isnon-responsive according to the RECIST (Response Evaluation Criteria InSolid Tumours) criteria or according to the irRECIST (immune-relatedResponse Evaluation Criteria In Solid Tumours) criteria.

According to some embodiments, the therapeutic combination of theinvention is for treating or preventing cancer in a subject in which aninhibitor of the invention or the bacterial composition alone cannotprovide effective treatment or prevention of cancer in the subject.According to some embodiments, an effective treatment of cancer in asubject comprises at least one of reducing tumour size, reducing tumourgrowth and/or preventing metastasis to an extent which will result incomplete or partial remission of the cancer in the subject.

According to some embodiments, the therapeutic combination of theinvention is capable of reducing tumour size and/or reducing tumourgrowth and/or preventing metastasis and/or preventing angiogenesis to ahigher extent than an inhibitor of the invention or the bacterialcomposition alone.

According to some embodiments, the therapeutic combination of theinvention is for treating cancer in a subject, such that there iscomplete remission of cancer in the subject, preferably in a shortertime frame than that achieved using treatment with the inhibitor of theinvention or the bacterial composition alone.

The invention also provides a composition comprising an inhibitorselected from the group consisting of Nivolumab, BGB-A137, cemiplimab,PDR001, camrelizumab, BCD-100, IBI308, AGEN2034, INCSHR1210, MEDI0680,JS001/PD1, CC-90006, BI 754091, JNJ-63723283, PF-06801591, GLS-010,AB122, Sym021, MGA012, LZMO09, genolimzumab, AK105, AB122, PF-06801591,PF-06688992 and TSR-042, for use in a method of treating or preventingcancer in a subject that had previously received administration of acomposition comprising a bacterial strain of the species Enterococcusgallinarum, preferably the strain deposited under accession number NCIMB42488.

The invention also provides a composition comprising a bacterial strainof the species Enterococcus gallinarum, preferably the strain depositedunder accession number NCIMB 42488, for use in a method of treating orpreventing cancer in a subject diagnosed as requiring treatment with aninhibitor selected from the group consisting of Nivolumab, BGB-A137,cemiplimab, PDR001, camrelizumab, BCD-100, IBI308, AGEN2034, INCSHR1210,MEDI0680, JS001/PD1, CC-90006, BI 754091, JNJ-63723283, PF-06801591,GLS-010, AB122, Sym021, MGA012, LZMO09, genolimzumab, AK105, AB122,PF-06801591, PF-06688992 and TSR-042.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A: Mouse model of breast cancer—tumor volume.

FIG. 1B: Upper panel: Area of necrosis in EMT6 tumours (Untreated n=6,Vehicle n=6, MRx0518 n=8). Lower panel: Percentage of dividing cells inEMT6 tumours. P=0.019 (Untreated n=4, total number cells counted=37201,Vehicle n=6, total number of cells counted=64297, MRx0518 n=6, totalnumber cells counted=33539).

FIG. 1C: Mouse model of breast cancer—infiltrating immune cells. Scatterplots represent cell counts of different immune markers from individualanimals from each treatment group.

FIG. 1D: Mouse model of breast cancer—Cytokine production in tumourlysates. Columns represent the mean pg/mL of total protein from eachtreatment group. *p<0.05 between groups using one-way ANOVA followed byDunnett's multiple comparisons test.

FIG. 1E: Mouse model of breast cancer—Cytokine production in bloodplasma. Columns represent the mean pg/mL from each treatment group(+/−SEM).

FIG. 1F: Representative images of ileum cryosections from vehicle,MRx0518 and CTLA-4-treated mice immuno-labelled with antibodies againstCD8α (lower panels) and counter-stained with DAPI (upper panels).

FIG. 1G: Plot quantifying animal study subsets with more than 3 CD8α+cells per field taken from the ileum crypt region of mice treated withvehicle, MRx0518 or CTLA-4.

FIG. 2 : Mouse model of lung cancer—tumour volume.

FIG. 3A: Mouse model of liver cancer—liver weight.

FIG. 3B: Mouse model of kidney cancer—tumour volume.

FIG. 4A: Cytokine levels (pg/ml) in immature dendritic cells (Nobacteria).

FIG. 4B: Cytokine levels (pg/ml) in immature dendritic cells after theaddition of LPS.

FIG. 4C: Cytokine levels (pg/ml) in immature dendritic cells after theaddition of MRX518.

FIG. 4D: Cytokine levels (pg/ml) in immature dendritic cells after theaddition of MRX518 and LPS.

FIG. 5A: Cytokine levels in THP-1 cells (No bacteria).

FIG. 5B: Cytokine levels in THP-1 cells after addition of bacterialsediment.

FIG. 5C: Cytokine levels in THP-1 cells after the addition of MRX518alone or in combination with LPS.

FIG. 6 : Bar graph depicting percentage of proliferating CD8+ cellsfollowing various treatments (NCD—No Cell Division, 1RCD—One CellDivision, 2RCD—Two Cell Divisions, 3RCD—Three Cell Divisions, 4RCD—FourCell Divisions).

FIG. 7A: A schematic representation of the treatment schedule of thedifferent groups used in Example 6 described herein below.

FIG. 7B: Mean tumour volume in mice bearing a tumour formed by EMT-6cells. The mice were either untreated or treated with a YCFA vehicle(Vehicle), MRx518 bacteria in YCFA medium (MRx518), an anti-PD1 antibody(RMP1-14) and YCFA medium (Anti-PD1), an anti-CTLA-4 antibody and YCFAmedium (Anti-CTLA-4), a combination of MRx518 and the anti-PD1 antibodyor a combination of MRx518 and the anti-CTLA-4 antibody.

DISCLOSURE OF THE INVENTION

Bacterial Strains

The compositions of the invention comprise a bacterial strain of thespecies Enterococcus gallinarum. The examples demonstrate that atherapeutic combination comprising bacteria of this species is usefulfor treating or preventing cancer.

According to some embodiments, provided herein is a therapeuticcombination for use in a method of treating or preventing cancer in asubject, wherein said therapeutic combination comprises:

-   -   (a) a composition comprising a bacterial strain of the species        Enterococcus gallinarum; and    -   (b) an inhibitor selected from the group consisting of        Nivolumab, BGB-A137, cemiplimab, PDR001, camrelizumab, BCD-100,        IBI308, AGEN2034, INCSHR1210, MEDI0680, JS001/PD1, CC-90006, BI        754091, JNJ-63723283, PF-06801591, GLS-010, AB122, Sym021,        MGA012, LZMO09, genolimzumab, AK105, AB122, PF-06801591,        PF-06688992 and TSR-042

According to some embodiments, a composition comprising a bacterialstrain that has a 16s rRNA sequence that is at least 95% identical tothe 16s rRNA sequence of a bacterial strain of Enterococcus gallinarummay be used in the therapeutic combination and method of the presentinvention. According to certain embodiments, the invention also providesa composition comprising a bacterial strain that has a 16s rRNA sequencethat is at least 95% identical to SEQ ID NO: 2 for use in treating orpreventing cancer in combination with an inhibitor of the invention. Insome embodiments, the bacterial strain in the composition is not ofEnterococcus gallinarum, but is a closely related strain.

In certain embodiments, the composition of the invention comprises abacterial strain that has a 16s rRNA sequence that is at least 95%identical to SEQ ID NO: 2, for example which is a Enterococcusgallinarum, and does not contain any other bacterial genus. In certainembodiments, the composition of the invention comprises a single strainof a bacterial strain that has a 16s rRNA sequence that is at least 95%identical to SEQ ID NO: 2, for example, which is an Enterococcusgallinarum, and does not contain any other bacterial strain or species.

Enterococcus gallinarum forms coccoid cells, mostly in pairs or shortchains. It is nonmotile and colonies on blood agar or nutrient agar arecircular and smooth. Enterococcus gallinarum reacts with Lancefieldgroup D antisera. The type strain of Enterococcus gallinarum isF87/276=PB21=ATCC 49573=CCUG 18658=CIP 103013=JCM 8728=LMG 13129=NBRC100675=NCIMB 702313 (formerly NCDO 2313)=NCTC 12359 [17]. The GenBankaccession number for a 16S rRNA gene sequence of Enterococcus gallinarumis AF039900 (disclosed herein as SEQ ID NO:1). An exemplary Enterococcusgallinarum strain is described in [17].

All microorganism deposits were made under the terms of the BudapestTreaty and thus viability of the deposit is assured. Maintenance of aviable culture is assured for 30 years from the date of deposit. Duringthe pendency of the application, access to the deposit will be affordedto one determined by the Commissioner of the United States Patent andTrademark Office to be entitled thereto. All restrictions on theavailability to the public of the deposited microorganisms will beirrevocably removed upon the granting of a patent for this application.The deposit will be maintained for a term of at least thirty (30) yearsfrom the date of the deposit or for the enforceable life of the patentor for a period of at least five (5) years after the most recent requestfor the furnishing of a sample of the deposited material, whichever islongest. The deposit will be replaced should it become necessary due toinviability, contamination or loss of capability to function in themanner described in the specification.

The Enterococcus gallinarum bacterium deposited under accession numberNCIMB 42488 was tested in the Examples and is also referred to herein asstrain MRX518. References to MRX518 and MRx0518 are usedinterchangeably. A 16S rRNA sequence for the MRX518 strain that wastested is provided in SEQ ID NO:2. Strain MRX518 was deposited with theinternational depositary authority NCIMB, Ltd. (Ferguson Building,Aberdeen, AB21 9YA, Scotland) by 4D Pharma Research Ltd. (Life SciencesInnovation Building, Aberdeen, AB25 2ZS, Scotland) on 16 Nov. 2015 as“Enterococcus sp” and was assigned accession number NCIMB 42488.

The genome of strain MRX518 comprises a chromosome and plasmid. Achromosome sequence for strain MRX518 is provided in SEQ ID NO:3 ofWO2017/085520. A plasmid sequence for strain MRX518 is provided in SEQID NO:4 of WO2017/085520. These sequences were generated using thePacBio RS II platform.

Bacterial strains closely related to the strain tested in the examplesare also expected to be effective for treating or preventing cancer inthe therapeutic combination of the invention. In certain embodiments,the bacterial strain for use in the therapeutic combination of theinvention has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%,99%, 99.5% or 99.9% identical to the 16s rRNA sequence of a bacterialstrain of Enterococcus gallinarum. Preferably, the bacterial strain foruse in the therapeutic combination of the invention has a 16s rRNAsequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%identical to SEQ ID NO:1 or 2. Preferably, the sequence identity is toSEQ ID NO:2. Preferably, the bacterial strain for use in the therapeuticcombination of the invention has the 16s rRNA sequence represented bySEQ ID NO:2.

Bacterial strains that are biotypes of the bacterium deposited underaccession number 42488 are also expected to be effective for treating orpreventing cancer in the context of the therapeutic combination of theinvention. A biotype is a closely related strain that has the same orvery similar physiological and biochemical characteristics.

Strains that are biotypes of the bacterium deposited under accessionnumber NCIMB 42488 and that are suitable for use in the therapeuticcombination of the invention may be identified by sequencing othernucleotide sequences for the bacterium deposited under accession numberNCIMB 42488. For example, substantially the whole genome may besequenced and a biotype strain for use in the therapeutic combination ofthe invention may have at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%sequence identity across at least 80% of its whole genome (e.g. acrossat least 85%, 90%, 95% or 99%, or across its whole genome). For example,in some embodiments, a biotype strain has at least 98% sequence identityacross at least 98% of its genome or at least 99% sequence identityacross 99% of its genome. Other suitable sequences for use inidentifying biotype strains may include hsp60 or repetitive sequencessuch as BOX, ERIC, (GTG)₅, or REP or [18]. Biotype strains may havesequences with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequenceidentity to the corresponding sequence of the bacterium deposited underaccession number NCIMB 42488. In some embodiments, a biotype strain hasa sequence with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%sequence identity to the corresponding sequence of strain MRX518deposited as NCIMB 42488 and comprises a 16S rRNA sequence that is atleast 99% identical (e.g. at least 99.5% or at least 99.9% identical) toSEQ ID NO:2. In some embodiments, a biotype strain has a sequence withat least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity tothe corresponding sequence of strain MRX518 deposited as NCIMB 42488 andhas the 16S rRNA sequence of SEQ ID NO:2.

In certain embodiments, the bacterial strain for use in the therapeuticcombination of the invention has a chromosome with sequence identity toSEQ ID NO:3 of WO2017/085520. In preferred embodiments, the bacterialstrain for use in the therapeutic combination of the invention has achromosome with at least 90% sequence identity (e.g. at least 92%, 94%,95%, 96%, 97%, 98%, 99% or 100% sequence identity) to SEQ ID NO:3 ofWO2017/085520 across at least 60% (e.g. at least 65%, 70%, 75%, 80%,85%, 95%, 96%, 97%, 98%, 99% or 100%) of SEQ ID NO:3 of WO2017/085520.For example, the bacterial strain for use in the therapeutic combinationof the invention may have a chromosome with at least 90% sequenceidentity to SEQ ID NO:3 of WO2017/085520 across 70% of SEQ ID NO:3 ofWO2017/085520, or at least 90% sequence identity to SEQ ID NO:3 ofWO2017/085520 across 80% of SEQ ID NO:3 of WO2017/085520, or at least90% sequence identity to SEQ ID NO:3 of WO2017/085520 across 90% of SEQID NO:3 of WO2017/085520, or at least 90% sequence identity to SEQ IDNO:3 of WO2017/085520 across 100% of SEQ ID NO:3 of WO2017/085520, or atleast 95% sequence identity to SEQ ID NO:3 of WO2017/085520 across 70%of SEQ ID NO:3 of WO2017/085520, or at least 95% sequence identity toSEQ ID NO:3 of WO2017/085520 across 80% of SEQ ID NO:3 of WO2017/085520,or at least 95% sequence identity to SEQ ID NO:3 of WO2017/085520 across90% of SEQ ID NO:3 of WO2017/085520, or at least 95% sequence identityto SEQ ID NO:3 of WO2017/085520 across 100% of SEQ ID NO:3 ofWO2017/085520, or at least 98% sequence identity to SEQ ID NO:3 ofWO2017/085520 across 70% of SEQ ID NO:3 of WO2017/085520, or at least98% sequence identity to SEQ ID NO:3 of WO2017/085520 across 80% of SEQID NO:3 of WO2017/085520, or at least 98% sequence identity to SEQ IDNO:3 of WO2017/085520 across 90% of SEQ ID NO:3 of WO2017/085520, or atleast 98% identity to SEQ ID NO:3 of WO2017/085520 across 95% of SEQ IDNO:3 of WO2017/085520, or at least 98% sequence identity to SEQ ID NO:3of WO2017/085520 across 100% of SEQ ID NO:3 of WO2017/085520, or atleast 99.5% sequence identity to SEQ ID NO:3 of WO2017/085520 across 90%of SEQ ID NO:3 of WO2017/085520, or at least 99.5% identity to SEQ IDNO:3 of WO2017/085520 across 95% of SEQ ID NO:3 of WO2017/085520, or atleast 99.5% identity to SEQ ID NO:3 of WO2017/085520 across 98% of SEQID NO:3 of WO2017/085520, or at least 99.5% sequence identity to SEQ IDNO:3 of WO2017/085520 across 100% of SEQ ID NO:3 of WO2017/085520.

In certain embodiments, the bacterial strain for use in the therapeuticcombination of the invention has a plasmid with sequence identity to SEQID NO:4 of WO2017/085520. In preferred embodiments, the bacterial strainfor use in the therapeutic combination of the invention has a plasmidwith at least 90% sequence identity (e.g. at least 92%, 94%, 95%, 96%,97%, 98%, 99% or 100% sequence identity) to SEQ ID NO:4 of WO2017/085520across at least 60% (e.g. at least 65%, 70%, 75%, 80%, 85%, 95%, 96%,97%, 98%, 99% or 100%) of SEQ ID NO:4 of WO2017/085520. For example, thebacterial strain for use in the therapeutic combination of the inventionmay have a plasmid with at least 90% sequence identity to SEQ ID NO:4 ofWO2017/085520 across 70% of SEQ ID NO:4 of WO2017/085520, or at least90% sequence identity to SEQ ID NO:4 of WO2017/085520 across 80% of SEQID NO:4 of WO2017/085520, or at least 90% sequence identity to SEQ IDNO:4 of WO2017/085520 across 90% of SEQ ID NO:4 of WO2017/085520, or atleast 90% sequence identity to SEQ ID NO:4 of WO2017/085520 across 100%of SEQ ID NO:4 of WO2017/085520, or at least 95% sequence identity toSEQ ID NO:4 of WO2017/085520 across 70% of SEQ ID NO:4 of WO2017/085520,or at least 95% sequence identity to SEQ ID NO:4 of WO2017/085520 across80% of SEQ ID NO:4 of WO2017/085520, or at least 95% sequence identityto SEQ ID NO:4 of WO2017/085520 across 90% of SEQ ID NO:4 ofWO2017/085520, or at least 95% sequence identity to SEQ ID NO:4 ofWO2017/085520 across 100% of SEQ ID NO:4 of WO2017/085520, or at least98% sequence identity to SEQ ID NO:4 of WO2017/085520 across 70% of SEQID NO:4 of WO2017/085520, or at least 98% sequence identity to SEQ IDNO:4 of WO2017/085520 across 80% of SEQ ID NO:4 of WO2017/085520, or atleast 98% sequence identity to SEQ ID NO:4 of WO2017/085520 across 90%of SEQ ID NO:4 of WO2017/085520, or at least 98% sequence identity toSEQ ID NO:4 of WO2017/085520 across 100% of SEQ ID NO:4 ofWO2017/085520.

In certain embodiments, the bacterial strain for use in the therapeuticcombination of the invention has a chromosome with sequence identity toSEQ ID NO:3 of WO2017/085520 and a plasmid with sequence identity to SEQID NO:4 of WO2017/085520.

In certain embodiments, the bacterial strain for use in the therapeuticcombination of the invention has a chromosome with sequence identity toSEQ ID NO:3 of WO2017/085520, for example as described above, and a 16SrRNA sequence with sequence identity to any of SEQ ID NO:1 or 2, forexample as described above, preferably with a 16s rRNA sequence that isat least 99% identical to SEQ ID NO: 2, more preferably which comprisesthe 16S rRNA sequence of SEQ ID NO:2, and optionally comprises a plasmidwith sequence identity to SEQ ID NO:4 of WO2017/085520, as describedabove.

In certain embodiments, the bacterial strain for use in the therapeuticcombination of the invention has a chromosome with sequence identity toSEQ ID NO:3 of WO2017/085520, for example as described above, andoptionally comprises a plasmid with sequence identity to SEQ ID NO:4 ofWO2017/085520, as described above, and is effective for treating orpreventing cancer.

In certain embodiments, the bacterial strain for use in the therapeuticcombination of the invention has a chromosome with sequence identity toSEQ ID NO:3 of WO2017/085520, for example as described above, and a 16SrRNA sequence with sequence identity to any of SEQ ID NOs: 1 or 2, forexample as described above, and optionally comprises a plasmid withsequence identity to SEQ ID NO:4 of WO2017/085520, as described above,and is effective for treating or preventing cancer.

In certain embodiments, the bacterial strain for use in the therapeuticcombination of the invention has a 16s rRNA sequence that is at least99%, 99.5% or 99.9% identical to the 16s rRNA sequence represented bySEQ ID NO: 2 (for example, which comprises the 16S rRNA sequence of SEQID NO:2) and a chromosome with at least 95% sequence identity to SEQ IDNO:3 of WO2017/085520 across at least 90% of SEQ ID NO:3 ofWO2017/085520, and optionally comprises a plasmid with sequence identityto SEQ ID NO:4 of WO2017/085520, as described above, and which iseffective for treating or preventing cancer.

In certain embodiments, the bacterial strain for use in the therapeuticcombination of the invention has a 16s rRNA sequence that is at least99%, 99.5% or 99.9% identical to the 16s rRNA sequence represented bySEQ ID NO: 2 (for example, which comprises the 16S rRNA sequence of SEQID NO:2) and a chromosome with at least 98% sequence identity (e.g. atleast 99% or at least 99.5% sequence identity) to SEQ ID NO:3 ofWO2017/085520 across at least 98% (e.g. across at least 99% or at least99.5%) of SEQ ID NO:3 of WO2017/085520, and optionally comprises aplasmid with sequence identity to SEQ ID NO:4 of WO2017/085520, asdescribed above, and which is effective for treating or preventingcancer.

In certain embodiments, the bacterial strain for use in the therapeuticcombination of the invention is a Enterococcus gallinarum and has a 16srRNA sequence that is at least 99%, 99.5% or 99.9% identical to the 16srRNA sequence represented by SEQ ID NO: 2 (for example, which comprisesthe 16S rRNA sequence of SEQ ID NO:2) and a chromosome with at least 98%sequence identity (e.g. at least 99% or at least 99.5% sequenceidentity) to SEQ ID NO:3 of WO2017/085520 across at least 98% (e.g.across at least 99% or at least 99.5%) of SEQ ID NO:3 of WO2017/085520,and optionally comprises a plasmid with sequence identity to SEQ ID NO:4of WO2017/085520, as described above, and which is effective fortreating or preventing cancer.

Alternatively, strains that are biotypes of the bacterium depositedunder accession number NCIMB 42488 and that are suitable for use in thetherapeutic combination of the invention may be identified by using theaccession number NCIMB 42488 deposit and restriction fragment analysisand/or PCR analysis, for example by using fluorescent amplified fragmentlength polymorphism (FAFLP) and repetitive DNA element (rep)-PCRfingerprinting, or protein profiling, or partial 16S or 23s rDNAsequencing. In preferred embodiments, such techniques may be used toidentify other Enterococcus gallinarum strains.

In certain embodiments, strains that are biotypes of the bacteriumdeposited under accession number NCIMB 42488 and that are suitable foruse in the therapeutic combination of the invention are strains thatprovide the same pattern as the bacterium deposited under accessionnumber NCIMB 42488 when analysed by amplified ribosomal DNA restrictionanalysis (ARDRA), for example when using Sau3AI restriction enzyme (forexemplary methods and guidance see, for example, [19]). Alternatively,biotype strains are identified as strains that have the samecarbohydrate fermentation patterns as the bacterium deposited underaccession number NCIMB 42488. In some embodiments, the carbohydratefermentation pattern is determined using the API 50 CHL panel(bioMérieux). In some embodiments, the bacterial strain used in thetherapeutic combination of the invention is:

-   -   (i) positive for fermentation of at least one of (e.g. at least        2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or all        of): L-arabinose, D-ribose, D-xylose, D-galactose, D-glucose,        D-fructose, D-mannose, N-acetylglucosamine, amygdalin, arbutin,        salicin, D-cellobiose, D-maltose, sucrose, D-trehalose,        gentiobiose, D-tagatose and potassium gluconate; and/or    -   (ii) intermediate for fermentation of at least one of (e.g. at        least 2, 3, 4 or all of): D-mannitol, Methyl-αD-glycopyranoside,        D-lactose, starch, and L-fucose;        -   preferably as determined by API 50 CHL analysis (preferably            using the API 50 CHL panel from bioMérieux).

Other Enterococcus gallinarum strains that are useful in thecompositions and methods of the invention, such as biotypes of thebacterium deposited under accession number NCIMB 42488, may beidentified using any appropriate method or strategy, including theassays described in the examples. For instance, strains for use in thetherapeutic combination of the invention may be identified by culturingin anaerobic YCFA and/or administering the bacteria to the type IIcollagen-induced arthritis mouse model and then assessing cytokinelevels. In particular, bacterial strains that have similar growthpatterns, metabolic type and/or surface antigens to the bacteriumdeposited under accession number NCIMB 42488 may be useful in thetherapeutic combination of the invention. A useful strain will havecomparable immune modulatory activity to the NCIMB 42488 strain. Inparticular, a biotype strain will elicit comparable effects on thecancer disease models to the effects shown in the Examples, which may beidentified by using the culturing and administration protocols describedin the Examples. According to some embodiments, a biotype strain thatmay be used in the therapeutic combination of the invention is a strainwhich is able to elicit comparable effects on the cancer disease modelsshown in the Examples when administered in the therapeutic combinationor method of the invention.

In some embodiments, the bacterial strain used in the therapeuticcombination of the invention is:

-   -   (i) Positive for at least one of (e.g. at least 2, 3, 4, 5, 6, 7        or all of): mannose fermentation, glutamic acid decarboxylase,        arginine arylamidase, phenylalanine arylamidase, pyroglutamic        acid arylamidase, tyrosine arylamidase, histidine arylamidase        and serine arylamidase; and/or    -   (ii) Intermediate for at least one of (e.g. at least 2 or all        of): β-galactosidase-6-phosphate, β-glucosidase and        N-acetyl-β-glucosaminidase; and/or    -   (iii) Negative for at least one of (e.g. at least 2, 3, 4, 5, 6        or all of): Raffinose fermentation, Proline arylamidase, Leucyl        glycine arylamidase, Leucine arylamidase, Alanine arylamidase,        Glycine arylamidase and Glutamyl glutamic acid arylamidase,

preferably as determined by an assay of carbohydrate, amino acid andnitrate metabolism, and optionally an assay of alkaline phosphataseactivity, more preferably as determined by Rapid ID 32A analysis(preferably using the Rapid ID 32A system from bioMérieux).

In some embodiments, the bacterial strain used in the therapeuticcombination of the invention is:

-   -   (i) Negative for at least one of (e.g. at least 2, 3, or all 4        of) glycine arylamidase, raffinose fermentation, proline        arylamidase, and leucine arylamidase, for example, as determined        by an assay of carbohydrate, amino acid and nitrate metabolism,        preferably as determined by Rapid ID 32A analysis (preferably        using the Rapid ID 32A system from bioMérieux); and/or    -   (ii) Intermediate positive for fermentation of L-fucose,        preferably as determined by API 50 CHL analysis (preferably        using the API 50 CHL panel from bioMérieux).

In some embodiments, the bacterial strain used in the therapeuticcombination of the invention is an extracellular ATP producer, forexample one which produces 6-6.7 ng/μl (for example, 6.1-6.6 ng/μl or6.2-6.5 ng/μl or 6.33±0.10 ng/μl) of ATP as measured using the ATP AssayKit (Sigma-Aldrich, MAK190). Bacterial extracellular ATP can havepleiotropic effects including activation of T cell-receptor mediatedsignalling (Schenk et al., 2011), promotion of intestinal Th17 celldifferentiation (Atarashi et al., 2008) and induction of secretion ofthe pro-inflammatory mediator IL-1β by activating the NLRP3 inflammasome(Karmarkar et al., 2016). Accordingly, a bacterial strain which is anextracellular ATP producer is useful for treating or preventing cancerin the context of the therapeutic combination and method of theinvention.

In some embodiments, the bacterial strain for use in the therapeuticcombination of the invention comprises one or more of the followingthree genes: Mobile element protein; Xylose ABC transporter, permeasecomponent; and FIG00632333: hypothetical protein. For example, incertain embodiments, the bacterial strain for use in the therapeuticcombination of the invention comprises genes encoding Mobile elementprotein and Xylose ABC transporter, permease component; Mobile elementprotein and FIG00632333: hypothetical protein; Xylose ABC transporter,permease component and FIG00632333: hypothetical protein; or Mobileelement protein, Xylose ABC transporter, permease component, andFIG00632333: hypothetical protein.

A particularly preferred strain of the therapeutic combination of theinvention is the Enterococcus gallinarum strain deposited underaccession number NCIMB 42488. This is the exemplary MRX518 strain testedin the examples and shown to be effective for treating disease. Theinvention provides, according to some embodiments, a bacterialcomposition as part of the therapeutic combination of the invention,comprising a cell of the Enterococcus gallinarum strain deposited underaccession number NCIMB 42488, or a derivative thereof. A derivative ofthe strain deposited under accession number NCIMB 42488 may be adaughter strain (progeny) or a strain cultured (subcloned) from theoriginal.

A derivative of a strain of the composition comprised in the therapeuticcombination of the invention may be modified, for example at the geneticlevel, without ablating the biological activity. In particular, aderivative strain of the therapeutic combination of the invention istherapeutically active. A derivative strain will have comparable immunemodulatory activity to the original NCIMB 42488 strain. In particular, aderivative strain will elicit comparable effects on the cancer diseasemodels when combined with an inhibitor of the invention to the effectsshown in the Examples, which may be identified by using the culturingand administration protocols described in the Examples. A derivative ofthe NCIMB 42488 strain will generally be a biotype of the NCIMB 42488strain.

References to cells of the Enterococcus gallinarum strain depositedunder accession number NCIMB 42488 encompass any cells that have thesame safety and therapeutic efficacy characteristics as the strainsdeposited under accession number NCIMB 42488, and such cells areencompassed by the therapeutic combination of the invention. Thus, insome embodiments, reference to cells of the Enterococcus gallinarumstrain deposited under accession number NCIMB 42488 refers only to theMRX518 strain deposited under NCIMB 42488 and does not refer to abacterial strain that was not deposited under NCIMB 42488. In someembodiments, reference to cells of the Enterococcus gallinarum straindeposited under accession number NCIMB 42488 refers to cells that havethe same safety and therapeutic efficacy characteristics as the strainsdeposited under accession number NCIMB 42488, but which are not thestrain deposited under NCIMB 42488.

In preferred embodiments, the bacterial strains in the compositions ofthe invention are viable and capable of partially or totally colonisingthe intestine.

Treating Cancer

In preferred embodiments, the therapeutic combinations of the inventionare for use in treating or preventing cancer. The examples demonstratethat administration of the therapeutic combinations of the invention canlead to a reduction in tumour growth.

In certain embodiments, treatment with the therapeutic combinations ofthe invention results in a reduction in tumour size or a reduction intumour growth. In certain embodiments, the therapeutic combinations ofthe invention are for use in reducing tumour size or reducing tumourgrowth. The therapeutic combinations of the invention may be effectivefor reducing tumour size or growth. In certain embodiments, thetherapeutic combinations of the invention are for use in patients withsolid tumours. In certain embodiments, the therapeutic combinations ofthe invention are for use in reducing or preventing angiogenesis in thetreatment of cancer. The therapeutic combinations of the invention mayhave an effect on the immune or inflammatory systems, which have centralroles in angiogenesis. In certain embodiments, the therapeuticcombinations of the invention are for use in preventing metastasis.

In certain embodiments, the therapeutic combinations of the inventionare for use in treating or preventing breast cancer. The examplesdemonstrate that the therapeutic combinations of the invention may beeffective for treating breast cancer. In certain embodiments, thetherapeutic combinations of the invention are for use in reducing tumoursize, reducing tumour growth, or reducing angiogenesis in the treatmentof breast cancer. In preferred embodiments the cancer is mammarycarcinoma. In preferred embodiments the cancer is stage IV breastcancer.

In certain embodiments, the therapeutic combinations of the inventionare for use in treating or preventing lung cancer. The examplesdemonstrate that the therapeutic combinations of the invention may beeffective for treating lung cancer. In certain embodiments, thetherapeutic combinations of the invention are for use in reducing tumoursize, reducing tumour growth, or reducing angiogenesis in the treatmentof lung cancer. In preferred embodiments the cancer is lung carcinoma.

In certain embodiments, the therapeutic combinations of the inventionare for use in treating or preventing liver cancer. The examplesdemonstrate that the therapeutic combinations of the invention may beeffective for treating liver cancer. In certain embodiments, thetherapeutic combinations of the invention are for use in reducing tumoursize, reducing tumour growth, or reducing angiogenesis in the treatmentof liver cancer. In preferred embodiments the cancer is hepatoma(hepatocellular carcinoma).

In certain embodiments, the therapeutic combinations of the inventionare for use in treating or preventing colon cancer. The examplesdemonstrate that the therapeutic combinations of the invention have aneffect on colon cancer cells and may be effective for treating coloncancer. In certain embodiments, the therapeutic combinations of theinvention are for use in reducing tumour size, reducing tumour growth,or reducing angiogenesis in the treatment of colon cancer. In preferredembodiments the cancer is colorectal adenocarcinoma.

In certain embodiments, the therapeutic combinations of the inventionare for use in treating or preventing kidney cancer (also referred toherein as renal cancer). The examples demonstrate that the therapeuticcombinations of the invention have an effect on renal cancer cells andmay be effective for treating renal cancer. In certain embodiments, thetherapeutic combinations of the invention are for use in reducing tumoursize, reducing tumour growth, or reducing angiogenesis in the treatmentof renal cancer. In preferred embodiments the cancer is renal cellcarcinoma or transitional cell carcinoma.

In certain embodiments, the therapeutic combinations of the inventionare for use in treating or preventing melanoma. According to someembodiments, the therapeutic combinations of the invention have aneffect on melanocytes and may be effective for treating melanoma. Incertain embodiments, the therapeutic combinations of the invention arefor use in reducing tumour size, reducing tumour growth, or reducingangiogenesis in the treatment of melanoma.

In some embodiments, the cancer is of the intestine. In someembodiments, the cancer is of a part of the body which is not theintestine. In some embodiments, the cancer is not cancer of theintestine. In some embodiments, the cancer is not colorectal cancer. Insome embodiments, the cancer is not cancer of the small intestine. Insome embodiments, the treating or preventing occurs at a site other thanat the intestine. In some embodiments, the treating or preventing occursat the intestine and also at a site other than at the intestine.

In certain embodiments, the therapeutic combinations of the inventionare for use in treating or preventing carcinoma. The examplesdemonstrate that the therapeutic combinations of the invention may beeffective for treating numerous types of carcinoma. In certainembodiments, the therapeutic combinations of the invention are for usein treating or preventing non-immunogenic cancer. The examplesdemonstrate that the therapeutic combinations of the invention may beeffective for treating non-immunogenic cancers.

The therapeutic effects of the bacterial compositions of the inventionon cancer, in the context of the therapeutic combinations of theinvention, may be mediated by a pro-inflammatory mechanism. Examples 2,4 and 5 demonstrate that the expression of a number of pro-inflammatorycytokines may be increased following administration of MRX518.Inflammation can have a cancer-suppressive effect [20] andpro-inflammatory cytokines such as TNFα are being investigated as cancertherapies [21]. The up-regulation of genes such as TNF shown in theexamples may indicate that the bacterial compositions of the inventionmay be useful for treating cancer via a similar mechanism. Theup-regulation of CXCR3 ligands (CXCL9, CXCL10) and IFNγ-inducible genes(IL-32) may indicate that the bacterial compositions of the inventionelicit an IFNγ-type response. IFNγ is a potent macrophage-activatingfactor that can stimulate tumirocidal activity [22], and CXCL9 andCXCL10, for example, also have anti-cancer effects [23-25]. Therefore,in certain embodiments, the bacterial compositions of the invention,when used in the context of the therapeutic combination of theinvention, are for use in promoting inflammation in the treatment ofcancer. In preferred embodiments, the compositions of the invention,when used in the context of the therapeutic combination of theinvention, are for use in promoting Th1 inflammation in the treatment ofcancer. Th1 cells produce IFNγ and have potent anti-cancer effects [20].In certain embodiments, the compositions of the invention, when used inthe context of the therapeutic combination of the invention, are for usein treating an early-stage cancer, such as a cancer that has notmetastasized, or a stage 0 or stage 1 cancer. Promoting inflammation maybe more effective against early-stage cancers [20]. In certainembodiments, the compositions of the invention, when used in the contextof the therapeutic combination of the invention, are for use inpromoting inflammation to enhance the effect of an inhibitor of theinvention. In certain embodiments, the treatment or prevention of cancercomprises increasing the level of expression of one or more cytokines.For example, in certain embodiments, the treatment or prevention ofcancer comprises increasing the level of expression of one or more ofIL-1β, IL-6 and TNF-α, for example, IL-1β and IL-6, IL-1β and TNF-α,IL-6 and TNF-α or all three of IL-1β, IL-6 and TNF-α. Increases inlevels of expression of any of IL-1β, IL-6 and TNF-α are known to beindicative of efficacy in treatment of cancer.

Examples 4 and 5 demonstrate that when a bacterial strain as describedherein is used in combination with lipopolysaccharide (LPS), there is asynergistic increase in IL-1β. LPS is known to elicit a pro-inflammatoryeffect. Thus, in certain embodiments, the treatment or prevention ofcancer comprises using a bacterial strain as described herein incombination with an agent that upregulates IL-1β. In certainembodiments, the treatment or prevention of cancer comprises using abacterial strain as described herein in combination with LPS.Accordingly, the therapeutic combination of the invention mayadditionally comprise an agent that upregulates IL-1β. Accordingly, thebacterial composition of the invention may additionally comprise LPS.

In certain embodiments, the therapeutic combinations of the inventionare for use in treating a patient that has previously receivedchemotherapy. In certain embodiments, the therapeutic combinations ofthe invention are for use in treating a patient that has not tolerated achemotherapy treatment. The therapeutic combinations of the inventionmay be particularly suitable for such patients. In other embodiments,the therapeutic combinations of the invention are for use in treating acancer patient who was non responsive to a prior treatment with animmune checkpoint inhibitor. In other embodiments, the therapeuticcombinations of the invention are for use in treating a cancer patientwho was non responsive to a prior treatment with a PD-1 inhibitor.Without wishing to be bound by theory or mechanism, it is believed thatthe bacterial composition of the invention is able to stimulate thesubject's immune system through a different mechanism to that ofinhibitors of the invention, thus providing a complementary mechanism totreat patients which are non-responsive to immune checkpoint inhibitors.

According to some embodiments, treatment of cancer using the therapeuticcombination of the invention results is more effective than using aninhibitor of the invention alone as measured by the RECIST (ResponseEvaluation Criteria In Solid Tumours) criteria or the irRECIST(immune-related Response Evaluation Criteria In Solid Tumours) criteria.According to some embodiments, treatment of cancer using the therapeuticcombination of the invention results is more effective than using thebacterial composition alone as measured by the RECIST (ResponseEvaluation Criteria In Solid Tumours) criteria or the irRECIST(immune-related Response Evaluation Criteria In Solid Tumours) criteria.According to some embodiment, treatment of cancer using the therapeuticcombination of the invention results in synergistic clinical effects ascompared to treatment with an inhibitor of the invention alone or thebacterial composition alone, as measured by the RECIST (ResponseEvaluation Criteria In Solid Tumours) criteria or the irRECIST(immune-related Response Evaluation Criteria In Solid Tumours) criteria.

In certain embodiments, the therapeutic combinations of the inventionare for preventing relapse. The bacterial compositions, in the contextof the therapeutic combinations of the invention, may be suitable forlong-term administration. In certain embodiments, the therapeuticcombinations of the invention are for use in preventing progression ofcancer.

In certain embodiments, the therapeutic combinations of the inventionare for use in treating non-small-cell lung carcinoma (NSCLC). Incertain embodiments, the therapeutic combinations of the invention arefor use in treating small-cell lung carcinoma. In certain embodiments,the therapeutic combinations of the invention are for use in treatingsquamous-cell carcinoma. In certain embodiments, the therapeuticcombinations of the invention are for use in treating adenocarcinoma. Incertain embodiments, the therapeutic combinations of the invention arefor use in treating glandular tumors, carcinoid tumors, orundifferentiated carcinomas.

In certain embodiments, the therapeutic combinations of the inventionare for use in treating hepatoblastoma, cholangiocarcinoma,cholangiocellular cystadenocarcinoma or liver cancer resulting from aviral infection.

In certain embodiments, the therapeutic combinations of the inventionare for use in treating invasive ductal carcinoma, ductal carcinoma insitu or invasive lobular carcinoma.

In further embodiments, the therapeutic combinations of the inventionare for use in treating or preventing acute lymphoblastic leukemia(ALL), acute myeloid leukemia, adrenocortical carcinoma, basal-cellcarcinoma, bile duct cancer, bladder cancer, bone tumor,osteosarcoma/malignant fibrous histiocytoma, brainstem glioma, braintumor, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma,ependymoma, medulloblastoma, supratentorial primitive neuroectodermaltumors, breast cancer, bronchial adenomas/carcinoids, Burkitt'slymphoma, carcinoid tumor, cervical cancer, chronic lymphocyticleukemia, chronic myelogenous leukemia, chronic myeloproliferativedisorders, colon cancer, cutaneous T-cell lymphoma, endometrial cancer,ependymoma, esophageal cancer, Ewing's sarcoma, intraocular melanoma,retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinalcarcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor,glioma, childhood visual pathway and hypothalamic, Hodgkin lymphoma,melanoma, islet cell carcinoma, Kaposi sarcoma, renal cell cancer,laryngeal cancer, leukaemias, lymphomas, mesothelioma, neuroblastoma,non-Hodgkin lymphoma, oropharyngeal cancer, osteosarcoma, ovariancancer, pancreatic cancer, parathyroid cancer, pharyngeal cancer,pituitary adenoma, plasma cell neoplasia, prostate cancer, renal cellcarcinoma, retinoblastoma, sarcoma, testicular cancer, thyroid cancer,or uterine cancer.

According to some embodiments, the therapeutic combinations are for usein treating or preventing cancer selected from the group consisting of:melanoma, NSCLC, bladder cancer and head-and-neck cancer.

In certain embodiments, the therapeutic combinations of the inventioncomprises an additional anticancer agent. According to some embodiments,the additional anticancer agent is selected from: a targeted antibodyimmunotherapy, a CAR-T cell therapy, an oncolytic virus, or a cytostaticdrug.

Modes of Administration

Preferably, the bacterial compositions of the invention are to beadministered to the gastrointestinal tract in order to enable deliveryto and/or partial or total colonisation of the intestine with thebacterial strain of the invention. Generally, the bacterial compositionsof the invention are administered orally, but they may be administeredrectally, intranasally, or via buccal or sublingual routes.

In certain embodiments, the bacterial compositions of the invention maybe administered as a foam, as a spray or a gel.

In certain embodiments, the bacterial compositions of the invention maybe administered as a suppository, such as a rectal suppository, forexample in the form of a theobroma oil (cocoa butter), synthetic hardfat (e.g. suppocire, witepsol), glycero-gelatin, polyethylene glycol, orsoap glycerin composition.

In certain embodiments, the bacterial composition of the invention isadministered to the gastrointestinal tract via a tube, such as anasogastric tube, orogastric tube, gastric tube, jejunostomy tube (Jtube), percutaneous endoscopic gastrostomy (PEG), or a port, such as achest wall port that provides access to the stomach, jejunum and othersuitable access ports.

The bacterial compositions of the invention may be administered once, orthey may be administered sequentially as part of a treatment regimen. Incertain embodiments, the bacterial compositions of the invention are tobe administered daily.

In certain embodiments of the invention, treatment according to theinvention is accompanied by assessment of the patient's gut microbiota.Treatment may be repeated if delivery of and/or partial or totalcolonisation with the strain of the bacterial composition of theinvention is not achieved such that efficacy is not observed, ortreatment may be ceased if delivery and/or partial or total colonisationis successful and efficacy is observed. According to some embodiments,the bacterial composition of the invention is administered to thesubject prior to first administration with the inhibitor of thetherapeutic combination of the invention. According to some embodiments,the subject's gut microbiota is assessed after administration of thebacterial composition and before first administration of the inhibitorof the invention, such that the inhibitor of the invention isadministered only after delivery and/or partial or total colonisationwith the strain of the bacterial strain in the composition is achieved.In certain embodiments, the therapeutic combination of the invention maybe administered to a pregnant animal, for example a mammal such as ahuman in order to reduce the likelihood of cancer developing in herchild in utero and/or after it is born.

The therapeutic combination of the invention may be administered to apatient that has been diagnosed with cancer, or that has been identifiedas being at risk of a cancer. The therapeutic combination may also beadministered as a prophylactic measure to prevent the development ofcancer in a healthy patient.

The therapeutic combination of the invention may be administered to apatient that has been identified as having an abnormal gut microbiota.For example, the patient may have reduced or absent colonisation byEnterococcus gallinarum.

The bacterial compositions of the invention may be administered as afood product, such as a nutritional supplement.

Generally, the therapeutic combinations of the invention are for thetreatment of humans, although they may be used to treat animalsincluding monogastric mammals such as poultry, pigs, cats, dogs, horsesor rabbits. The therapeutic combinations of the invention may be usefulfor enhancing the growth and performance of animals. If the bacterialcomposition is administered to animals, oral gavage may be used.

According to some embodiments, the inhibitor of the invention isadministered intravenously. According to some embodiments, the inhibitorof the invention which is administered intravenously is in a compositionwhich optionally further comprises at least one pharmaceuticallycompatible carrier or excipient. According to some embodiments, theinhibitor of the invention is administered intravenously every aboutone, two, three or four weeks, preferably every three weeks.

According to some embodiments, the bacterial composition and theinhibitor of the therapeutic combination of the invention areadministered using different administration routes. According to someembodiments, the bacterial composition is administered orally whereasthe inhibitor of the therapeutic combination of the invention isadministered using a different route. According to some embodiments, theinhibitor of the therapeutic combination is administered intravenouslywhereas the bacterial composition is administered orally.

According to some embodiments, the bacterial composition is administeredto the subject prior to a first administration of inhibitor of theinvention to the subject. According to some embodiments, the bacterialcomposition is administered to the subject prior to a firstadministration of the inhibitor of the invention to the subject; whereinthe bacterial composition is administered until delivery and/or partialor total colonisation with the strain of the bacterial strain in thecomposition is achieved. According to some embodiments, the bacterialcomposition is administered to the subject prior to a firstadministration of the inhibitor of the invention to the subject; whereinthe bacterial composition is administered until sufficient modulation ofbiomarkers occurs such that the inhibitor of the invention is capable oftreating a cancer patient who was previously non-responsive to inhibitortreatment. According to some embodiments, the bacterial composition isadministered to the subject for at least one, two, three or four weeksprior to first administration of the inhibitor of the invention.According to some embodiments, the bacterial composition is administeredto the subject for about two weeks prior to first administration of theinhibitor of the invention. According to some embodiments, the bacterialcomposition is administered to the subject for at least one, two, threeor four weeks prior to first administration of the inhibitor of theinvention and is not administered to the subject in parallel toadministration of the inhibitor of the invention.

According to some embodiments, the first administration of the bacterialcomposition in the therapeutic combination of the invention is prior tothe first administration of the inhibitor of the invention. According tosome embodiments, the first administration of the inhibitor of theinvention occurs no more than about 1, 2, 3, 4, 5, 6 or 7 days followingadministration of the bacterial composition.

According to some embodiments of the method and therapeutic combinationof the invention, the bacterial composition is administered to thesubject at least partially in parallel to administration of theinhibitor of the invention to the subject. According to someembodiments, the bacterial composition is administered to the subjectfor a first time period, followed by administration of the inhibitor ofthe invention to the subject for a second time period; wherein thebacterial composition is optionally further administered to the subjectfor at least part of said second time period, optionally all through thesecond time period. According to certain embodiments, the bacterialcomposition is administered to the subject for a first time period, suchas, but not limited to, for about two weeks, followed by administrationof the inhibitor of the invention to the subject for a second timeperiod, such as, but not limited to, for about three weeks. According tocertain embodiments, the bacterial composition is administered to thesubject for a first time period, such as, but not limited to, for abouttwo weeks, followed by administration of the inhibitor of the inventionto the subject for a second time period, such as, but not limited to,for about three weeks; wherein the bacterial composition is furtheradministered to the subject for at least part of said second timeperiod, preferably all through the second time period.

According to some embodiments, the bacterial composition and theinhibitor of the invention are not administered at the same frequency.In a non-limiting example, the inhibitor of the invention isadministered intravenously every three weeks, whereas the bacterialcomposition is administered orally every day or every other day.According to some embodiments, the bacterial composition is administeredto the subject for a first time period, followed by administration ofthe inhibitor of the invention to the subject for a second time period;wherein the bacterial composition is optionally further administered tothe subject for at least part of said second time period; and whereinthe frequency of administration of the bacterial composition isdifferent in the first time period and second time period.

Bacterial Compositions of the Therapeutic Combination of the Invention

Generally, the composition comprised in the therapeutic combination ofthe invention comprises bacteria. In preferred embodiments of theinvention, the bacterial composition is formulated in freeze-dried form.For example, the bacterial composition of the invention may comprisegranules or gelatin capsules, for example hard gelatin capsules,comprising a bacterial strain of the invention.

Preferably, the bacterial composition of the invention compriseslyophilised bacteria. Lyophilisation of bacteria is a well-establishedprocedure and relevant guidance is available in, for example, references[26-28].

Alternatively, the bacterial composition of the invention may comprise alive, active bacterial culture.

In some embodiments, the bacterial strain in the bacterial compositionof the invention has not been inactivated, for example, has not beenheat-inactivated. In some embodiments, the bacterial strain in thebacterial composition of the invention has not been killed, for example,has not been heat-killed. In some embodiments, the bacterial strain inthe bacterial composition of the invention has not been attenuated, forexample, has not been heat-attenuated. For example, in some embodiments,the bacterial strain in the bacterial composition of the invention hasnot been killed, inactivated and/or attenuated. For example, in someembodiments, the bacterial strain in the bacterial composition of theinvention is live. For example, in some embodiments, the bacterialstrain in the bacterial composition of the invention is viable. Forexample, in some embodiments, the bacterial strain in the bacterialcomposition of the invention is capable of partially or totallycolonising the intestine. For example, in some embodiments, thebacterial strain in the bacterial composition of the invention is viableand capable of partially or totally colonising the intestine.

In some embodiments, the bacterial composition comprises a mixture oflive bacterial strains and bacterial strains that have been killed.

In preferred embodiments, the bacterial composition of the therapeuticcombination of the invention is encapsulated to enable delivery of thebacterial strain to the intestine. Encapsulation protects the bacterialcomposition from degradation until delivery at the target locationthrough, for example, rupturing with chemical or physical stimuli suchas pressure, enzymatic activity, or physical disintegration, which maybe triggered by changes in pH. Any appropriate encapsulation method maybe used. Exemplary encapsulation techniques include entrapment within aporous matrix, attachment or adsorption on solid carrier surfaces,self-aggregation by flocculation or with cross-linking agents, andmechanical containment behind a microporous membrane or a microcapsule.Guidance on encapsulation that may be useful for preparing compositionsof the invention is available in, for example, references [29] and [30].

The bacterial composition may be administered orally and may be in theform of a tablet, capsule or powder. Encapsulated products are preferredbecause Enterococcus gallinarum are anaerobes. Other ingredients (suchas vitamin C, for example), may be included as oxygen scavengers andprebiotic substrates to improve the delivery and/or partial or totalcolonisation and survival in vivo. Alternatively, the probioticcomposition of the invention may be administered orally as a food ornutritional product, such as milk or whey based fermented dairy product,or as a pharmaceutical product.

The bacterial composition may be formulated as a probiotic.

A bacterial composition of the invention includes a therapeuticallyeffective amount of a bacterial strain of the invention. Atherapeutically effective amount of a bacterial strain is sufficient toexert a beneficial effect upon a patient. A therapeutically effectiveamount of a bacterial strain may be sufficient to result in delivery toand/or partial or total colonisation of the patient's intestine.

A suitable daily dose of the bacteria, for example for an adult human,may be from about 1×10³ to about 1×10¹¹ colony forming units (CFU); forexample, from about 1×10⁷ to about 1×10¹⁰ CFU; in another example fromabout 1×10⁶ to about 1×10¹⁰ CFU.

In certain embodiments, the bacterial composition contains the bacterialstrain in an amount of from about 1×10⁶ to about 1×10¹¹ CFU/g, respectto the weight of the composition; for example, from about 1×10⁸ to about1×10¹⁰ CFU/g. The dose may be, for example, 1 g, 3 g, 5 g, and 10 g.

A probiotic, such as the bacterial composition of the invention, mayoptionally be combined with at least one suitable prebiotic compound. Aprebiotic compound is usually a non-digestible carbohydrate such as anoligo- or polysaccharide, or a sugar alcohol, which is not degraded orabsorbed in the upper digestive tract. Known prebiotics includecommercial products such as inulin and transgalacto-oligosaccharides.

In certain embodiments, the probiotic bacterial composition of thepresent invention includes a prebiotic compound in an amount of fromabout 1 to about 30% by weight, respect to the total weight composition,(e.g. from 5 to 20% by weight). Carbohydrates may be selected from thegroup consisting of: fructo-oligosaccharides (or FOS), short-chainfructo-oligosaccharides, inulin, isomalt-oligosaccharides, pectins,xylo-oligosaccharides (or XOS), chitosan-oligosaccharides (or COS),beta-glucans, arable gum modified and resistant starches, polydextrose,D-tagatose, acacia fibers, carob, oats, and citrus fibers. In oneaspect, the prebiotics are the short-chain fructo-oligosaccharides (forsimplicity shown herein below as FOSs-c.c); said FOSs-c.c. are notdigestible carbohydrates, generally obtained by the conversion of thebeet sugar and including a saccharose molecule to which three glucosemolecules are bonded.

The bacterial compositions of the invention may comprisepharmaceutically acceptable excipients or carriers. Examples of suchsuitable excipients may be found in the reference [31]. Acceptablecarriers or diluents for therapeutic use are well known in thepharmaceutical art and are described, for example, in reference [32].Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol and the like. Examplesof suitable diluents include ethanol, glycerol and water. The choice ofpharmaceutical carrier, excipient or diluent can be selected with regardto the intended route of administration and standard pharmaceuticalpractice. The pharmaceutical compositions may comprise as, or inaddition to, the carrier, excipient or diluent any suitable binder(s),lubricant(s), suspending agent(s), coating agent(s), solubilisingagent(s). Examples of suitable binders include starch, gelatin, naturalsugars such as glucose, anhydrous lactose, free-flow lactose,beta-lactose, corn sweeteners, natural and synthetic gums, such asacacia, tragacanth or sodium alginate, carboxymethyl cellulose andpolyethylene glycol. Examples of suitable lubricants include sodiumoleate, sodium stearate, magnesium stearate, sodium benzoate, sodiumacetate, sodium chloride and the like. Preservatives, stabilizers, dyesand even flavouring agents may be provided in the pharmaceuticalcomposition. Examples of preservatives include sodium benzoate, sorbicacid and esters of p-hydroxybenzoic acid. Antioxidants and suspendingagents may be also used.

The bacterial compositions of the invention may be formulated as a foodproduct. For example, a food product may provide nutritional benefit inaddition to the therapeutic effect of the invention, such as in anutritional supplement. Similarly, a food product may be formulated toenhance the taste of the composition of the invention or to make thecomposition more attractive to consume by being more similar to a commonfood item, rather than to a pharmaceutical composition. In certainembodiments, the composition of the invention is formulated as amilk-based product. The term “milk-based product” means any liquid orsemi-solid milk- or whey-based product having a varying fat content. Themilk-based product can be, e.g., cow's milk, goat's milk, sheep's milk,skimmed milk, whole milk, milk recombined from powdered milk and wheywithout any processing, or a processed product, such as yoghurt, curdledmilk, curd, sour milk, sour whole milk, butter milk and other sour milkproducts. Another important group includes milk beverages, such as wheybeverages, fermented milks, condensed milks, infant or baby milks;flavoured milks, ice cream; milk-containing food such as sweets.

In certain embodiments, the bacterial compositions of the inventioncontain a single bacterial strain or species and do not contain anyother bacterial strains or species. Such bacterial compositions maycomprise only de minimis or biologically irrelevant amounts of otherbacterial strains or species. Such bacterial compositions may be aculture that is substantially free from other species of organism. Thus,in some embodiments, the bacterial composition of the therapeuticcombination comprises one or more strains from the species Enterococcusgallinarum, and does not contain bacteria from any other species orcomprises only de minimis or biologically irrelevant amounts of bacteriafrom another species.

In some embodiments, the bacterial compositions of the inventioncomprise more than one bacterial strain or species. For example, in someembodiments, the bacterial compositions of the invention comprise morethan one strain from within the same species (e.g. more than 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or 45 strains), and,optionally, do not contain bacteria from any other species. In someembodiments, the bacterial compositions of the invention comprise lessthan 50 strains from within the same species (e.g. less than 45, 40, 35,30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4 or 3 strains), and, optionally,do not contain bacteria from any other species. In some embodiments, thebacterial compositions of the invention comprise 1-40, 1-30, 1-20, 1-19,1-18, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40,2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or 31-50 strains fromwithin the same species and, optionally, do not contain bacteria fromany other species. In some embodiments, the bacterial compositions ofthe invention comprise more than one species from within the same genus(e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 23, 25,30, 35 or 40 species), and, optionally, do not contain bacteria from anyother genus. In some embodiments, the bacterial compositions of theinvention comprise less than 50 species from within the same genus (e.g.less than 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, 7, 6, 5, 4 or 3species), and, optionally, do not contain bacteria from any other genus.In some embodiments, the bacterial compositions of the inventioncomprise 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5,1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15,16-25, or 31-50 species from within the same genus and, optionally, donot contain bacteria from any other genus. The bacterial composition foruse in the combination of the invention may comprise any combination ofthe foregoing.

In some embodiments, the bacterial composition comprises a microbialconsortium. For example, in some embodiments, the bacterial compositioncomprises the bacterial strain having a 16s rRNA sequence that is atleast 95% identical to SEQ ID NO:2, for example, which is anEnterococcus gallinarum, as part of a microbial consortium. For example,in some embodiments, the bacterial strain is present in the bacterialcomposition in combination with one or more (e.g. at least 2, 3, 4, 5,10, 15 or 20) other bacterial strains from other genera with which itcan live symbiotically in vivo in the intestine. For example, in someembodiments, the bacterial composition comprises a bacterial strainhaving a 16s rRNA sequence that is at least 95% identical to SEQ IDNO:2, for example, which is an Enterococcus gallinarum, in combinationwith a bacterial strain from a different genus. In some embodiments, themicrobial consortium comprises two or more bacterial strains obtainedfrom a faeces sample of a single organism, e.g. a human. In someembodiments, the microbial consortium is not found together in nature.For example, in some embodiments, the microbial consortium comprisesbacterial strains obtained from faeces samples of at least two differentorganisms. In some embodiments, the two different organisms are from thesame species, e.g. two different humans, e.g. two different humaninfants. In some embodiments, the two different organisms are an infanthuman and an adult human. In some embodiments, the two differentorganisms are a human and a non-human mammal.

In some embodiments, the bacterial composition of the inventionadditionally comprises a bacterial strain that has the same safety andtherapeutic efficacy characteristics as strain MRX518, but which is notMRX518 deposited as NCIMB 42488, or which is not an Enterococcusgallinarum.

In some embodiments, the bacterial strain for use in the bacterialcomposition is obtained from human infant faeces. In some embodiments inwhich the bacterial composition comprises more than one bacterialstrain, all of the bacterial strains are obtained from human infantfaeces or if other bacterial strains are present they are present onlyin de minimis amounts. The bacteria may have been cultured subsequent tobeing obtained from the human infant faeces and being used in thebacterial composition.

As mentioned above, in some embodiments, the one or more bacterialstrains having a 16s rRNA sequence that is at least 95% identical to SEQID NO:2, for example which is an Enterococcus gallinarum, is/are theonly therapeutically active agent(s) in the bacterial composition of theinvention. In some embodiments, the bacterial strain(s) in the bacterialcomposition is/are the only therapeutically active agent(s) in thecomposition.

The bacterial compositions for use in accordance with the invention mayor may not require marketing approval.

In certain embodiments, the invention provides the above bacterialcomposition, wherein said bacterial strain is lyophilised. In certainembodiments, the invention provides the above bacterial composition,wherein said bacterial strain is spray dried. In certain embodiments,the invention provides the above bacterial composition, wherein thebacterial strain is lyophilised or spray dried and wherein it is alive.

In certain embodiments, the invention provides the above bacterialcomposition, wherein the bacterial strain is lyophilised or spray driedand wherein it is viable. In certain embodiments, the invention providesthe above bacterial composition, wherein the bacterial strain islyophilised or spray dried and wherein it is capable of partially ortotally colonising the intestine. In certain embodiments, the inventionprovides the above bacterial composition, wherein the bacterial strainis lyophilised or spray dried and wherein it is viable and capable ofpartially or totally colonising the intestine.

In some cases, the lyophilised or spray dried bacterial strain isreconstituted prior to administration. In some cases, the reconstitutionis by use of a diluent described herein.

The bacterial compositions of the invention can comprisepharmaceutically acceptable excipients, diluents or carriers.

In certain embodiments, the bacterial composition is a pharmaceuticalcomposition comprising: a bacterial strain as used in the invention; anda pharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in combination with an inhibitor of theinvention; and wherein the disorder is breast cancer. In preferredembodiments the cancer is mammary carcinoma. In preferred embodimentsthe cancer is stage IV breast cancer.

In certain embodiments, the bacterial composition is a pharmaceuticalcomposition comprising: a bacterial strain as used in the invention; anda pharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in combination with an inhibitor of theinvention; and wherein the disorder is lung cancer. In preferredembodiments the cancer is lung carcinoma.

In certain embodiments, the bacterial composition is a pharmaceuticalcomposition comprising: a bacterial strain as used in the invention; anda pharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in combination with an inhibitor of theinvention; and wherein the disorder is liver cancer. In preferredembodiments the cancer is hepatoma (hepatocellular carcinoma).

In certain embodiments, the bacterial composition is a pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in combination with an inhibitor of theinvention; and wherein the disorder is colon cancer. In preferredembodiments the cancer is colorectal adenocarcinoma.

In certain embodiments, the bacterial composition is a pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent;

wherein the bacterial strain is in an amount sufficient to treat adisorder when administered to a subject in combination with an inhibitorof the invention; and wherein the disorder is carcinoma.

In certain embodiments, the bacterial composition is a pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in combination with an inhibitor of theinvention; and wherein the disorder is a non-immunogenic cancer.

In certain embodiments, the bacterial composition is a pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in combination with an inhibitor of theinvention; and wherein the disorder is selected from the groupconsisting of non-small-cell lung carcinoma, small-cell lung carcinoma,squamous-cell carcinoma, adenocarcinoma, glandular tumors, carcinoidtumors undifferentiated carcinomas.

In certain embodiments, the bacterial composition is a pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent;

wherein the bacterial strain is in an amount sufficient to treat adisorder when administered to a subject in combination with an inhibitorof the invention; and wherein the disorder is selected from the groupconsisting of hepatoblastoma, cholangiocarcinoma, cholangiocellularcystadenocarcinoma or liver cancer resulting from a viral infection.

In certain embodiments, the bacterial composition is a pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in combination with an inhibitor of theinvention; and wherein the disorder is selected from the groupconsisting of invasive ductal carcinoma, ductal carcinoma in situ orinvasive lobular carcinoma.

In certain embodiments, the bacterial composition is a pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in combination with an inhibitor of theinvention; and wherein the disorder is selected from the groupconsisting of acute lymphoblastic leukemia (ALL), acute myeloidleukemia, adrenocortical carcinoma, basal-cell carcinoma, bile ductcancer, bladder cancer, bone tumor, osteosarcoma/malignant fibroushistiocytoma, brainstem glioma, brain tumor, cerebellar astrocytoma,cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma,supratentorial primitive neuroectodermal tumors, breast cancer,bronchial adenomas/carcinoids, Burkitt's lymphoma, carcinoid tumor,cervical cancer, chronic lymphocytic leukemia, chronic myelogenousleukemia, chronic myeloproliferative disorders, colon cancer, cutaneousT-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer,Ewing's sarcoma, intraocular melanoma, retinoblastoma, gallbladdercancer, gastric cancer, gastrointestinal carcinoid tumor,gastrointestinal stromal tumor (GIST), germ cell tumor, glioma,childhood visual pathway and hypothalamic, Hodgkin lymphoma, melanoma,islet cell carcinoma, Kaposi sarcoma, renal cell cancer, laryngealcancer, leukaemias, lymphomas, mesothelioma, neuroblastoma, non-Hodgkinlymphoma, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreaticcancer, parathyroid cancer, pharyngeal cancer, pituitary adenoma, plasmacell neoplasia, prostate cancer, renal cell carcinoma, retinoblastoma,sarcoma, testicular cancer, thyroid cancer, or uterine cancer.

In certain embodiments, the amount of the bacterial strain in thebacterial composition is from about 1×10³ to about 1×10¹¹ colony formingunits per gram with respect to a weight of the composition.

In certain embodiments, the bacterial composition is administered at adose of 1 g, 3 g, 5 g or 10 g.

In certain embodiments, the bacterial composition is administered by amethod selected from the group consisting of oral, rectal, subcutaneous,nasal, buccal, and sublingual.

In certain embodiments, the bacterial composition comprises a carrierselected from the group consisting of lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol and sorbitol.

In certain embodiments, the invention provides the bacterial compositioncomprises a diluent selected from the group consisting of ethanol,glycerol and water.

In certain embodiments, the bacterial composition comprises an excipientselected from the group consisting of starch, gelatin, glucose,anhydrous lactose, free-flow lactose, beta-lactose, corn sweetener,acacia, tragacanth, sodium alginate, carboxymethyl cellulose,polyethylene glycol, sodium oleate, sodium stearate, magnesium stearate,sodium benzoate, sodium acetate and sodium chloride.

In certain embodiments, the bacterial composition further comprises atleast one of a preservative, an antioxidant and a stabilizer.

In certain embodiments, the bacterial composition comprises apreservative selected from the group consisting of sodium benzoate,sorbic acid and esters of p-hydroxybenzoic acid.

In certain embodiments, when the bacterial composition is stored in asealed container at about 4.0 or about 25.0 and the container is placedin an atmosphere having 50% relative humidity, at least 80% of thebacterial strain as measured in colony forming units, remains after aperiod of at least about: 1 month, 3 months, 6 months, 1 year, 1.5years, 2 years, 2.5 years or 3 years.

In some embodiments, the bacterial composition of the invention isprovided in a sealed container. In some embodiments, the sealedcontainer is a sachet or bottle. In some embodiments, the bacterialcomposition of the invention is provided in a syringe.

The bacteria; composition may, in some embodiments, be provided as apharmaceutical formulation. For example, the bacterial composition maybe provided as a tablet or capsule. In some embodiments, the capsule isa gelatine capsule (“gel-cap”).

In some embodiments, the bacterial compositions of the invention areadministered orally. In some embodiments, the bacterial compositions ofthe inventions are formulated in a pharmaceutical formulation suitablefor oral administration. Oral administration may involve swallowing, sothat the compound enters the gastrointestinal tract, and/or buccal,lingual, or sublingual administration by which the compound enters theblood stream directly from the mouth.

Pharmaceutical formulations suitable for oral administration includesolid plugs, solid microparticulates, semi-solid and liquid (includingmultiple phases or dispersed systems) such as tablets; soft or hardcapsules containing multi- or nano-particulates, liquids (e.g. aqueoussolutions), emulsions or powders; lozenges (including liquid-filled);chews; gels; fast dispersing dosage forms; films; ovules; sprays; andbuccal/mucoadhesive patches.

In some embodiments the pharmaceutical formulation is an entericformulation, i.e. a gastro-resistant formulation (for example, resistantto gastric pH) that is suitable for delivery of the composition of theinvention to the intestine by oral administration. Enteric formulationsmay be particularly useful when the bacteria or another component of thecomposition is acid-sensitive, e.g. prone to degradation under gastricconditions.

In some embodiments, the enteric formulation comprises an entericcoating. In some embodiments, the formulation is an enteric-coateddosage form. For example, the formulation may be an enteric-coatedtablet or an enteric-coated capsule, or the like. The enteric coatingmay be a conventional enteric coating, for example, a conventionalcoating for a tablet, capsule, or the like for oral delivery. Theformulation may comprise a film coating, for example, a thin film layerof an enteric polymer, e.g. an acid-insoluble polymer.

In some embodiments, the enteric formulation is intrinsically enteric,for example, gastro-resistant without the need for an enteric coating.Thus, in some embodiments, the formulation is an enteric formulationthat does not comprise an enteric coating. In some embodiments, theformulation is a capsule made from a thermogelling material. In someembodiments, the thermogelling material is a cellulosic material, suchas methylcellulose, hydroxymethylcellulose orhydroxypropylmethylcellulose (HPMC). In some embodiments, the capsulecomprises a shell that does not contain any film forming polymer. Insome embodiments, the capsule comprises a shell and the shell compriseshydroxypropylmethylcellulose and does not comprise any film formingpolymer (e.g. see [33]). In some embodiments, the formulation is anintrinsically enteric capsule (for example, Vcaps® from Capsugel).

In some embodiments, the formulation is a soft capsule. Soft capsulesare capsules which may, owing to additions of softeners, such as, forexample, glycerol, sorbitol, maltitol and polyethylene glycols, presentin the capsule shell, have a certain elasticity and softness. Softcapsules can be produced, for example, on the basis of gelatine orstarch. Gelatine-based soft capsules are commercially available fromvarious suppliers. Depending on the method of administration, such as,for example, orally or rectally, soft capsules can have various shapes,they can be, for example, round, oval, oblong or torpedo-shaped. Softcapsules can be produced by conventional processes, such as, forexample, by the Scherer process, the Accogel process or the droplet orblowing process.

Culturing Methods

The bacterial strains for use in the present invention can be culturedusing standard microbiology techniques as detailed in, for example,references [34-36].

The solid or liquid medium used for culture may be YCFA agar or YCFAmedium. YCFA medium may include (per 100 ml, approximate values):Casitone (1.0 g), yeast extract (0.25 g), NaHCO₃ (0.4 g), cysteine (0.1g), K₂HPO₄ (0.045 g), KH₂PO₄ (0.045 g), NaCl (0.09 g), (NH₄)₂SO₄ (0.09g), MgSO₄·7H₂O (0.009 g), CaCl₂) (0.009 g), resazurin (0.1 mg), hemin (1mg), biotin (1 μg), cobalamin (1 μg), p-aminobenzoic acid (3 μg), folicacid (5 μg), and pyridoxamine (15 μg).

Bacterial strains for use in vaccine compositions

The inventors have identified that the bacterial strains of thebacterial composition of the invention are useful for treating orpreventing cancer when administered in combination with an inhibitorselected from the group consisting of Nivolumab, BGB-A137, cemiplimab,PDR001, camrelizumab, BCD-100, IBI308, AGEN2034, INCSHR1210, MEDI0680,JS001/PD1, CC-90006, BI 754091, JNJ-63723283, PF-06801591, GLS-010,AB122, Sym021, MGA012, LZMO09, genolimzumab, AK105, AB122, PF-06801591,PF-06688992 and TSR-042. This is likely to be a result of the effectthat the bacterial strains of the invention have on the host immunesystem. In certain embodiments, the bacterial strains are viable. Incertain embodiments, the bacterial strains are capable of partially ortotally colonising the intestine. In certain embodiments, the bacterialstrains are viable and capable of partially or totally colonising theintestine. In other certain embodiments, the bacterial strains may bekilled, inactivated or attenuated. In certain embodiments, the bacterialcompositions are for administration via injection, such as viasubcutaneous injection.

Inhibitors of the Invention

The therapeutic combination of the invention comprises at least oneinhibitor selected from the group consisting of Nivolumab, BGB-A137,cemiplimab, PDR001, camrelizumab, BCD-100, IBI308, AGEN2034, INCSHR1210,MEDI0680, JS001/PD1, CC-90006, BI 754091, JNJ-63723283, PF-06801591,GLS-010, AB122, Sym021, MGA012, LZMO09, genolimzumab, AK105, AB122,PF-06801591, PF-06688992 and TSR-042 (collectively referred to herein as‘inhibitors’, ‘inhibitors of the invention’ or ‘inhibitors of thetherapeutic combination’, or singly as ‘inhibitor, inhibitor of theinvention’, or ‘inhibitor of the therapeutic combination’). Thesecompounds inhibit immune checkpoints, thus enabling the body's immunesystem to attack cells that are recognized as the body's own cells,including cancer cells.

Nivolumab is marketed by Bristol-Myers Squibb under the commercial nameOPVIDO® and BGB-A137 is developed by BeiGene for the treatment ofvarious cancer types.

The term “antibody” refers to any form of antibody that exhibits thedesired biological activity, such as inhibiting binding of a ligand toits receptor, or inhibiting ligand-induced signaling of a receptor.Thus, “antibody” is used in the broadest sense and specifically covers,but is not limited to, monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, and multispecificantibodies (e.g., bispecific antibodies). According to some embodiments,an antibody (or antigen binding fragment thereof) used in the presentinvention is an isolated antibody.

The terms “antibody fragment”, “antigen binging fragment” and “antibodybinding fragment” used interchangeably throughout the application meanantigen-binding fragments, typically including at least a portion of theantigen binding or variable regions (e.g. one or more CDRs) of theparental antibody. An antibody fragment retains at least some of thebinding specificity of the parental antibody. Typically, an antibodyfragment retains at least 10% of the parental binding activity when thatactivity is expressed on a molar basis. Preferably, an antibody fragmentretains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of theparental antibody's binding affinity for the target. Examples ofantibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2,and Fv fragments; single-chain antibody molecules, e.g., sc-Fv.

The “Fab fragment” is comprised of one light chain and the CH1 andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule.

A “Fab′ fragment” contains one light chain and a portion of one heavychain that contains the V_(H) domain and the CH1 domain and also theregion between the CH1 and CH2 domains, such that an interchaindisulfide bond can be formed between the two heavy chains of two Fab′fragments to form a F(ab′)2 molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the CH1 and CH2domains, such that an interchain disulfide bond is formed between thetwo heavy chains A F(ab′)₂ fragment thus is composed of two Fab′fragments that are held together by a disulfide bond between the twoheavy chains.

The “Fv region” comprises the variable regions from both the heavy andlight chains, but lacks the constant regions.

A “single-chain Fv antibody” (or “scFv antibody”) refers to antibodyfragments comprising the VH and VL domains of an antibody, wherein thesedomains are present in a single polypeptide chain. Generally, the Fvpolypeptide further comprises a polypeptide linker between the V_(H) andV_(L) domains which enables the scFv to form the desired structure forantigen binding.

An “isolated” antibody is an antibody that has been separated and/orrecovered from a component of its natural environment. In someembodiments, the antibody will be purified to greater than 95% by weightof antibody as determined by the Lowry method, and most preferably morethan 99% by weight.

A “human antibody” is an antibody that possesses an amino acid sequencecorresponding to that of an antibody produced by a human. Thisdefinition specifically excludes a humanized antibody that comprisesnon-human antigen-binding residues.

A “chimeric” antibody refers to antibodies in which a portion of theheavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, so long as they exhibit the desired biologicalactivity.

“Humanized” forms of non-human (for example, murine) antibodies arechimeric antibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

The term “hypervariable region” as used herein, refers to the amino acidresidues of an antibody which are responsible for antigen-binding. Thehypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR”.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The term “monoclonal” indicates the character of the antibodyas being obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, monoclonal antibodiesmay be made by the hybridoma method, or may be made by recombinant DNAmethods. The monoclonal antibodies may also be isolated from phageantibody libraries.

The terms “specific binding” or “specifically bind” as used hereinrefers to a non-random association between two molecules, i.e., antibodyand antigen. According to some embodiments, the antibody or antigenbinding fragment thereof, via its antigen-binding domain, specificallybinds to the antigen with a binding affinity (Kd) of <10⁻⁵ M.Alternatively, the antibody or antigen binding fragment thereof, via itsantigen-binding domain, may bind to the antigen with a Kd of <10⁻⁶ M or<10⁻ M. Kd, as used herein, refers to the ratio of the dissociation rateto the association rate (k_(off)/k_(on)), and may be determined usingany suitable methods known in the art.

According to some embodiments, the inhibitor of the therapeuticcombination is administered systemically. According to some embodiments,the inhibitor of the invention is formulated for systemicadministration.

According to another embodiment, administration systemically is througha parenteral route. According to some embodiments, preparations of theinhibitor of the invention for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, or emulsions, eachrepresenting a separate embodiment of the present invention.

According to some embodiments, parenteral administration isadministration intravenously, intra-arterially, administering into ablood-vessel wall, intramuscularly, intraperitoneally, intradermally,intravitreally, transdermally or subcutaneously. Each of theabovementioned administration routes represents a separate embodiment ofthe present invention. According to some embodiments, the inhibitor ofthe therapeutic combination is administered intravenously.

According to some embodiments, systemic administration of the inhibitorof the invention is through injection. For administration throughinjection, the inhibitor of the invention may be formulated in anaqueous solution, for example in a physiologically compatible buffer,including, but not limited to, Hank's solution, Ringer's solution, orphysiological salt buffer. Formulations for injection may be presentedin unit dosage forms, for example, in ampoules, or in multi-dosecontainers with, optionally, an added preservative. Aqueous injectionsuspensions may contain substances that increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the activeingredients, to allow for the preparation of highly concentratedsolutions.

According to some embodiment, parenteral administration is performed bybolus injection. According to other embodiments, parenteraladministration is performed by continuous infusion. According to someembodiments, the inhibitor of the invention is delivered in a controlledrelease system and is formulated for intravenous infusion, implantableosmotic pump, transdermal patch, liposomes, or other modes ofadministration. In one embodiment, a pump is used. In yet anotherembodiment, a controlled release system can be placed in proximity tothe therapeutic target, thus requiring only a fraction of the systemicdose.

The therapeutic combination According to some embodiments, providedherein is the therapeutic combination of the invention for use in amethod of treating or preventing cancer in a subject. According to someembodiments, provided herein is the therapeutic combination of theinvention for use in a method of treating cancer in a subject.

According to some embodiments, cancer to be treated or prevented usingthe therapeutic combination of the invention is selected from the groupconsisting of: melanoma, non-small cell lung carcinoma, bladder cancerand head-and-neck cancer. According to some embodiments, cancer to betreated or prevented using the therapeutic combination of the inventionis selected from the group consisting of:

breast cancer, lung cancer, colon cancer and liver cancer.

According to some embodiments, treating cancer relates to at least oneof reducing tumour size or preventing tumour growth in a subject.According to some embodiments, the therapeutic combination or the methodof the invention is for use in at least one of: reducing tumour size,reducing tumour growth, preventing metastasis or preventing angiogenesisin a subject afflicted with cancer.

According to some embodiments, the therapeutic combination of theinvention comprises: (a) a composition comprising a bacterial strain ofthe species Enterococcus gallinarum; and (b) an inhibitor selected fromthe group consisting of Nivolumab, BGB-A137, cemiplimab, PDR001,camrelizumab, BCD-100, IBI308, AGEN2034, INCSHR1210, MEDI0680,JS001/PD1, CC-90006, BI 754091, JNJ-63723283, PF-06801591, GLS-010,AB122, Sym021, MGA012, LZMO09, genolimzumab, AK105, AB122, PF-06801591,PF-06688992 and TSR-042.

According to some embodiments, the bacterial composition of thetherapeutic combination does not contain bacteria from any other speciesother than Enterococcus gallinarum, or comprises only de minimis orbiologically irrelevant amounts of bacteria from another species.According to some embodiments, the bacterial composition of thetherapeutic combination contains only a single strain of the speciesEnterococcus gallinarum, and does not contain bacteria from any otherspecies or comprises only de minimis or biologically irrelevant amountsof bacteria from another species. According to some embodiments, thebacterial composition of the therapeutic combination comprises theEnterococcus gallinarum strain deposited under accession number NCIMB42488. According to some embodiments, the bacterial composition of thetherapeutic combination comprises a single strain of the Enterococcusgallinarum species, deposited under accession number NCIMB 42488, anddoes not contain bacteria from any other species or comprises only deminimis or biologically irrelevant amounts of bacteria from anotherspecies.

According to some embodiments, the inhibitor of the invention is in acomposition, possibly comprising at least one pharmaceuticallyacceptable carrier and/or excipient.

According to some embodiments, the therapeutic combination of theinvention comprises: (a) a composition comprising a bacterial strain ofthe species Enterococcus gallinarum, wherein the composition comprises asingle strain of the Enterococcus gallinarum species, deposited underaccession number NCIMB 42488, optionally wherein the composition doesnot contain bacteria from any other species or comprises only de minimisor biologically irrelevant amounts of bacteria from another species; and(b) an inhibitor selected from the group consisting of Nivolumab,BGB-A137, cemiplimab, PDR001, camrelizumab, BCD-100, IBI308, AGEN2034,INCSHR1210, MEDI0680, JS001/PD1, CC-90006, BI 754091, JNJ-63723283,PF-06801591, GLS-010, AB122, Sym021, MGA012, LZMO09, genolimzumab,AK105, AB122, PF-06801591, PF-06688992 and TSR-042.

Preferably, the therapeutic combination of the invention comprises: (a)a composition comprising the bacterial strain of the speciesEnterococcus gallinarum, deposited under accession number NCIMB 42488;and (b) an inhibitor selected from the group consisting of Nivolumab,BGB-A137, cemiplimab, PDR001, camrelizumab, BCD-100, IBI308, AGEN2034,INCSHR1210, MEDI0680, JS001/PD1, CC-90006, BI 754091, JNJ-63723283,PF-06801591, GLS-010, AB122, Sym021, MGA012, LZMO09, genolimzumab,AK105, AB122, PF-06801591, PF-06688992 and TSR-042. According to someembodiments, provided herein is a therapeutic combination for use in amethod of treating or preventing cancer in a subject, wherein thetherapeutic combination comprises: (a) a composition comprising thebacterial strain of the species Enterococcus gallinarum, deposited underaccession number NCIMB 42488; and (b) an inhibitor selected from thegroup consisting of Nivolumab, BGB-A137, cemiplimab, PDR001,camrelizumab, BCD-100, IBI308, AGEN2034, INCSHR1210, MEDI0680,JS001/PD1, CC-90006, BI 754091, JNJ-63723283, PF-06801591, GLS-010,AB122, Sym021, MGA012, LZMO09, genolimzumab, AK105, AB122, PF-06801591,PF-06688992 and TSR-042.

According to some embodiments, the therapeutic combination of theinvention comprises: (a) a composition comprising a bacterial strain ofthe species Enterococcus gallinarum; and (b) an inhibitor selected fromthe group consisting of Nivolumab, BGB-A137, cemiplimab, PDR001,camrelizumab, BCD-100, IBI308, AGEN2034, INCSHR1210, MEDI0680,JS001/PD1, CC-90006, BI 754091, JNJ-63723283, PF-06801591, GLS-010,AB122, Sym021, MGA012, LZMO09, genolimzumab, AK105, AB122, PF-06801591,PF-06688992 and TSR-042.

According to some embodiments, the therapeutic combination of theinvention comprises: (a) a composition comprising a bacterial strain ofthe species Enterococcus gallinarum, optionally the strain depositedunder accession number NCIMB 42488, optionally wherein the compositiondoes not contain bacteria from any other species and/or strains orcomprises only de minimis or biologically irrelevant amounts of bacteriafrom another species and/or strain; and (b) an inhibitor selected fromthe group consisting of Nivolumab, BGB-A137, cemiplimab, PDR001,camrelizumab, BCD-100, IBI308, AGEN2034, INCSHR1210, MEDI0680,JS001/PD1, CC-90006, BI 754091, JNJ-63723283, PF-06801591, GLS-010,AB122, Sym021, MGA012, LZMO09, genolimzumab, AK105, AB122, PF-06801591,PF-06688992 and TSR-042.

According to some embodiments, provided herein is a method for treatingand/or preventing cancer in a subject using any one of the therapeuticcombinations disclosed herein. According to some embodiments, thepresent invention provides any one of the therapeutic combinationsdisclosed herein for use in treating and/or preventing cancer in asubject.

General

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., references[37] and [38-44], etc.

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x is optional andmeans, for example, x+10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

References to a percentage sequence identity between two nucleotidesequences means that, when aligned, that percentage of nucleotides arethe same in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in section 7.7.18 of ref.[45]. A preferred alignment is determined by the Smith-Waterman homologysearch algorithm using an affine gap search with a gap open penalty of12 and a gap extension penalty of 2, BLOSUM matrix of 62. TheSmith-Waterman homology search algorithm is disclosed in ref [46].

Unless specifically stated, a process or method comprising numeroussteps may comprise additional steps at the beginning or end of themethod, or may comprise additional intervening steps. Also, steps may becombined, omitted or performed in an alternative order, if appropriate.

Various embodiments of the invention are described herein. It will beappreciated that the features specified in each embodiment may becombined with other specified features, to provide further embodiments.In particular, embodiments highlighted herein as being suitable, typicalor preferred may be combined with each other (except when they aremutually exclusive).

MODES FOR CARRYING OUT THE INVENTION Example 1— Efficacy of BacterialInocula in Mouse Models of Cancer Summary

This study tested the efficacy of compositions comprising bacterialstrains according to the invention in four tumor models.

Materials

Test substance—Bacterial strain #MRX518.

Reference substance—Anti-CTLA-4 antibody (clone: 9H10, catalog: BE0131,isotype: Syrian Hamster IgG1, Bioxcell).

Test and reference substances vehicles—Bacterial culture medium (Yeastextract, Casitone, Fatty Acid medium (YCFA)). Each day of injection tomice, antibody was diluted with PBS (ref: BE14-516F, Lonza, France).

Treatment doses—Bacteria: 2×10⁸ in 200 μL. The a-CTLA-4 was injected at10 mg/kg/inj. Anti-CTLA-4 was administered at a dose volume of 10mL/kg/adm (i.e. for one mouse weighing 20 g, 200 μL of test substancewill be administered) according to the most recent body weight of mice.

Routes of administration—Bacterial inoculum was administered by oralgavage (per os, PO) via a cannula. Cannulas were decontaminated everyday. Anti-CTLA-4 was injected into the peritoneal cavity of mice(Intraperitoneally, IP).

Culture conditions of bacterial strain—The culture conditions for thebacterial strain were as follows:

-   -   Pipette 10 mL of YCFA (from the prepared 10 mL E&O lab bottles)        into Hungate tubes    -   Seal the tubes and flush with CO₂ using a syringe input and        exhaust system    -   Autoclave the Hungate tubes    -   When cooled, inoculate the Hungate tubes with 1 mL of the        glycerol stocks    -   Place the tubes in a static 37° C. incubator for about 16 hours.    -   The following day, take 1 mL of this subculture and inoculate 10        mL of YCFA (pre-warmed flushed Hungate tubes again, all in        duplicate)    -   Place them in a static 37° C. incubator for 5 to 6 h

Cancer Cell Line and Culture Conditions—

The cell lines that were used are detailed in the table below:

Cell line Type Mouse strain Origin EMT-6 Breast carcinoma BALB/c ATCCLL/2 (LLC1) Lung carcinoma C57BL/6 ATCC CRL1642 Hepa1-6 Hepatocellularcarcinoma C57BL/6 IPSEN INNOVATION RENCA Renal adenocarcinoma BALB/cATCC

The EMT-6 cell line was established from a transplantable murine mammarycarcinoma that arose in a BALB/cCRGL mouse after implantation of ahyperplastic mammary alveolar nodule [47].

The LL/2 (LLC1) cell line was established from the lung of a C57BL mousebearing a tumor resulting from an implantation of primary Lewis lungcarcinoma [48].

The Hepa 1-6 cell line is a derivative of the BW7756 mouse hepatoma thatarose in a C57/L mouse [49].

Cell culture conditions—All cell lines were grown as monolayer at 37° C.in a humidified atmosphere (5% CO₂, 95% air). The culture medium andsupplement are indicated in the table below:

Cell line Culture medium Supplement EMT6 RPMI 1640 containing 2 mM 10%fetal bovine serum L-glutamine (ref: BE12-702F, (ref: #3302, Lonza)Lonza) LL/2 RPMI 1640 containing 2 mM 10% fetal bovine serum (LLC1)L-glutamine (ref: BE12-702F, (ref: #3302, Lonza) Lonza) Hepa1-6 DMEM(ref: 11960-044, Gibco) 10% fetal bovine serum (ref: #3302, Lonza) 2 mML-Glutamine penicillin-streptomycin (Sigma G-6784) RENCA DMEM 10% fetalbovine serum, 2 mM L-glutamine, 1 ug/ml puromycin

For experimental use, adherent tumor cells were detached from theculture flask by a 5 minute treatment with trypsin-versene (ref:BE17-161E, Lonza), in Hanks' medium without calcium or magnesium (ref:BE10-543F, Lonza) and neutralized by addition of complete culturemedium. The cells were counted in a hemocytometer and their viabilitywill be assessed by 0.25% trypan blue exclusion assay.

Use of Animals—

Healthy female Balb/C (BALB/cByJ) mice, of matching weight and age, wereobtained from CHARLES RIVER (L'Arbresles) for the EMT6 and RENCA modelexperiments.

Healthy female C57BL/6 (C57BL16J) mice, of matching weight and age, wereobtained from CHARLES RIVER (L'Arbresles) for the LL/2(LLC1) and theHepal-6 model experiments.

Animals were maintained in SPF health status according to the FELASAguidelines, and animal housing and experimental procedures according tothe French and European Regulations and NRC Guide for the Care and Useof Laboratory Animals were followed [50,51]. Animals were maintained inhousing rooms under controlled environmental conditions: Temperature:22±2° C., Humidity 55±10%, Photoperiod (12 h light/12 h dark), HEPAfiltered air, 15 air exchanges per hour with no recirculation. Animalenclosures were provided with sterile and adequate space with beddingmaterial, food and water, environmental and social enrichment (grouphousing) as described: 900 cm² cages (ref: green, Tecniplast) inventilated racks, Epicea bedding (SAFE), 10 kGy Irradiated diet (A04-10,SAFE), Complete food for immuno-competent rodents—R/M-H Extrudate, waterfrom water bottles.

Experimental Design and Treatments

Antitumor Activity, EMT6 Model

Treatment schedule—The start of first dosing was considered as D0. OnD0, non-engrafted mice were randomized according to their individualbody weight into groups of 9/8 using Vivo Manager® software(Biosystemes, Couternon, France). On D0, the mice received vehicle(culture medium) or bacterial strain. On D14, all mice were engraftedwith EMT-6 tumor cells as described below. On D24, mice from thepositive control group received anti-CTLA-4 antibody treatments.

The treatment schedule is summarized in the table below:

Treatment Group No. Animals Treatment Dose Route Schedule 1 8 Untreated— — — 2 8 Vehicle (media) — PO Q1Dx42 3 9 Bacterial strain #1 2 × 108 POQ1Dx42 (MRX518) bacteria 4 8 Anti-CTLA4 10 mg/kg IP TWx2

The monitoring of animals was performed as described below.

Induction of EMT6 tumors in animals—On D14, tumors were induced bysubcutaneous injection of 1×10⁶ EMT-6 cells in 200 μL RPMI 1640 into theright flank of mice.

Euthanasia—Each mouse was euthanized when it reached a humane endpointas described below, or after a maximum of 6 weeks post start of dosing.

Antitumor Activity, LL/2 (LLC1) Model

Treatment schedule—The start of first dosing was considered as D0. OnD0, non-engrafted mice were randomized according to their individualbody weight into 7 groups of 9/8 using Vivo Manager® software(Biosystemes, Couternon, France). On D0, the mice will received vehicle(culture medium) or bacterial strain. On D14, all mice were engraftedwith LL/2 tumor cells as described below. On D27, mice from the positivecontrol group received anti-CTLA-4 antibody treatments.

The treatment schedule is summarized in the table below:

Treatment Group No. Animals Treatment Dose Route Schedule 1 8 Untreated— — — 2 9 Vehicle (media) — PO Q1Dx42 3 9 Bacterial strain #1 2 × 10⁸ POQ1Dx42 (MRX518) bacteria 4 8 Anti-CTLA4 10 mg/kg IP TWx2

The monitoring of animals was performed as described below.

Induction of LL/2 (LLC1) tumors in animals—On D14, tumors were inducedby subcutaneous injection of 1×10⁶ LL/2 (LLC1) cells in 200 μL RPMI 1640into the right flank of mice.

Euthanasia—Each mouse was euthanized when it reached a humane endpointas described below, or after a maximum of 6 weeks post start of dosing.

Antitumor activity, Hepal-6 model

Treatment schedule—The start of first dosing was considered as D0. OnD0, non-engrafted mice were randomized according to their individualbody weight into 7 groups of 9 using Vivo Manager® software(Biosystemes, Couternon, France). On D0, the mice received vehicle(culture medium) or bacterial strain. On D14, all mice were engraftedwith Hepa 1-6 tumor cells as described below. On D16, mice from thepositive control group received anti-CTLA-4 antibody treatments.

The treatment schedule is summarized in the table below:

Treatment Group No. Animals Treatment Dose Route Schedule 1 9 Untreated— — — 2 9 Vehicle (media) — PO Q1Dx42 6 9 Bacterial strain #4 2 × 10⁸ POQ1Dx42 (MRX518) bacteria 7 9 Anti-CTLA4 10 mg/kg IP TWx2

The monitoring of animals was performed as described below.

Orthotopic induction of Hepa 1-6 tumor cells in animals by intrasplenicinjection—On D14, one million (1×10⁶) Hepa 1-6 tumor cells in 50 μL RPMI1640 medium were transplanted via intra-splenic injection into mice.Briefly, a small left subcostal flank incision was made and the spleenwas exteriorized. The spleen was exposed on a sterile gauze pad, andinjected under visual control with the cell suspension with a 27-gaugeneedle. After the cell inoculation, the spleen was excised.

Euthanasia—Each mouse was euthanized when it reached a humane endpointas described in section below, or after a maximum of 6 weeks post startof dosing.

Evaluation of tumor burden at euthanasia—At the time of termination,livers were collected and weighed.

Antitumor Activity, RENCA Model

Treatment schedule—The start of first dosing was considered as D0. OnD0, non-engrafted mice were randomized according to their individualbody weight into groups of 9 mice using Vivo Manager® software(Biosystemes, Couternon, France). On D0, the mice received vehicle(culture medium) or bacterial strain (2×10⁸ in 200 μL, PO). On D14, allmice were engrafted with RENCA tumour cells injected SC into the ventralsurface of the lower flank as described below. Treatment withanti-CTLA-4 (10 mg/kg, IP) and anti-PDL1 (clone 10F.9G2, 10 mg/kg) wasinitiated when tumours reached a volume of 50-70 mm3.

The treatment schedule is summarized in the table below:

Treatment Group No. Animals Treatment Dose Route Schedule 1 9 Untreated— — — 2 9 Vehicle — PO Q1Dx42 (media) 3 9 Bacterial strain 2 × 10⁸ POQ1Dx42 (MRX518) bacteria 4 9 Paclitaxel 15 mg/kg IP Q4D (every fourdays) 5 9 Anti-CTLA4 + 10 mg/kg + IP TWx2 Anti-PDL1 10 mg/kg

The monitoring of animals was performed as described below.

Orthotopic induction of RENCA tumor cells in animals by SC injection—OnD14, one million (1×10⁶) RENCA tumor cells in 50 μL RPMI 1640 mediumwere transplanted via SC injection into the ventral surface of the lowerflank of mice.

Euthanasia—Each mouse was euthanized when it reached a humane endpointas described in section below, or after a maximum of 6 weeks post startof dosing.

Evaluation of tumour burden at euthanasia—At the time of termination,tumours were collected and their volume evaluated.

Animal Monitoring

Clinical monitoring—The length and width of the tumour was measuredtwice a week with callipers and the volume of the tumour was estimatedby this formula [52]:

${{Tumor}{volume}} = \frac{{width}^{2} \times {length}}{2}$

Humane endpoints [53]: Signs of pain, suffering or distress: painposture, pain face mask, behaviour; Tumor exceeding 10% of normal bodyweight, but non-exceeding 2000 mm³; Tumors interfering with ambulationor nutrition; Ulcerated tumor or tissue erosion; 20% body weight lossremaining for 3 consecutive days; Poor body condition, emaciation,cachexia, dehydration; Prolonged absence of voluntary responses toexternal stimuli; Rapid laboured breathing, anaemia, significantbleeding; Neurologic signs: circling, convulsion, paralysis; Sustaineddecrease in body temperature; Abdominal distension.

Anaesthesia—Isoflurane gas anesthesia were used for all procedures:surgery or tumor inoculation, i.v. injections, blood collection.Ketamine and Xylazine anesthesia were used for stereotaxia surgicalprocedure.

Analgesia—Carprofen or multimodal carprofen/buprenorphine analgesiaprotocol were adapted to the severity of surgical procedure.Non-pharmacological care was provided for all painful procedures.Additionally, pharmacological care not interfering with studies (topictreatment) were provided at the recommendation of the attendingveterinarian.

Euthanasia—Euthanasia of animals was performed by gas anesthesiaover-dosage (Isoflurane) followed by cervical dislocation orexsanguination.

Results

Antitumor Activity, EMT6 Model

The results are shown in FIG. 1A. Treatment with the bacterial strain ofthe invention led to a clear reduction in tumour volume relative to boththe negative controls. The positive control also led to a reduction intumour volume, as would be expected.

To further elucidate the mechanisms through which MRx0518 conveys itstherapeutic effects in syngeneic tumour models, ex vivo analysis wasperformed on the syngeneic EMT6 tumour model studies. While tumourvolume is the primary measurement in preclinical oncology studies,tumours often consist of actively dividing tumour cells along with anecrotic core. To investigate whether MRx0518 treatment had influence onthe degree of necrosis found within EMT6 tumours, paraffin sections fromthe mid-belly region of the tumours were stained with Haematoxylin andEosin. MRx0518 treatment of a murine EMT6 breast carcinoma model showeda tendency towards increasing the cross-sectional area of necrosiswithin the tumour (FIG. 1B, upper panel). To investigate whether MRx0518treatment had influence on dividing cells within the tumour, paraffinsections from the mid-belly region of the tumours were stained with theproliferation protein Ki67, along with DAPI counter stain, to estimatethe percentage of cells dividing within the EMT6 tumour. MRx0518treatment of a murine EMT6 breast carcinoma model significantlydecreased the percentage of dividing cells seen within the tumour (FIG.1B, lower panel, P=0.019).

Immune Cell Populations

Further investigation of the tumour microenvironment was performedthrough flow cytometry of the tumour, to investigate the hypothesis thatthe MRx518 bacterial strain has the ability to regulate the immunesystem into inducing an anti-tumour effect. Tumours excised from thedifferent treatment groups were cut into pieces. One piece was subjectedto flow cytometry analysis. To assess the relative percentage of Tlymphocytes, present within the tumours, the following markers wereused: CD45, CD3, CD4, CD8, CD25 and FoxP3.

The flow cytometry data shows that the relative percentage oflymphocytes in tumours was slightly decreased in both the MRx0518 andanti-CTLA-4 treated groups, when compared respectively to vehicle orcontrol animals (FIG. 1C). Likewise, the relative percentage of CD4+cells appeared to be decreased in MRx0518 and anti-CTLA-4 treatedanimals, whilst the relative percentage of CD8+ cells followed anopposite trend in both groups, albeit with different magnitude. Therelative percentage of CD4+FoxP3+ cells was lower in the anti-CTLA-4treated group when compared to the slight decrease in MRx0518 treatedanimals; however, the reduction in the relative percentage of CD4+CD25+cells was noticeable only in the anti-CTLA-4 treated group. TheCD8+/FoxP3+ ratio showed a greater increase in the anti-CTLA-4 treatedgroup than in the MRx0518 animals. These data presented here supportsthe hypothesis that anti-CTLA-4 antibody targets regulatory T cells(Tregs) by reducing their cell numbers or attenuating their suppressiveactivity in tumour tissue, whilst suggesting a different mode of actionfor MRx0518.

Cytokine Production

An additional tumour piece was used for total protein extraction andsubsequent cytokine analysis, together with plasma samples. Proteinlevels of IL-10, CXCL1, CXCL2, CXCL10, IL-1B, IL-17A, GM-CSF, TNF-α,IL-12p70 and IFN-γ in the tumour microenvironment were analysed byMagPix technology. While IL-17A and GM-CSF were below levels ofdetection, all the other markers were expressed at reasonable levels(FIG. 1D). A significance difference was observed between the vehicleand anti-CTLA-4 group for IFN-γ. The production of the IL-10 andIL-12p70 immune markers seemed reduced following MRx518 treatmentcompared to the control treatments.

Cytokine levels were also assessed in blood plasma of the same animals.Protein levels of IL-23, IL-6, IL-10, VEGF, CXCL1, CXCL2, CXCL10, IL-2,IL-1B, IL-17A, GM-CSF, TNF-α, IL-12p70 and IFN-γ were analysed by MagPixtechnology. Overall, little cytokine production was detected in theblood plasma of animals either before tumour induction or at the end ofthe study (FIG. 1E). VEGF and CXCL10 were detected at substantiallevels, while IL-23, IL-6, IL-10, CXCL1 and CXCL2 were detected at lowlevels. IL-2, IL-1b, IL-17A, GM-CSF, TNF-α, IL-12p70 and IFN-γ were notdetected in the samples. MRx0518 significantly increased production ofIL-6 at Day 0. MRx0518 also seemed to increase IL-23 production. VEGFand CXCL10 were significantly downregulated in the anti-CLTA-4 group atDay 22. Similarly to the results shown for the immune cell populations,the differences in cytokine production in the tumour and plasma, betweenMRx518 and CTLA-4 suggests that each of them acts on a distinct andpotentially complementary mechanism.

Localisation of CD8α Positive Cells in the Ileum

10 μm cryo-sections of ileum were cut in cryostat (CM 1950 Leica),picked up onto poly-L Lysine slides. The sections were then air-driedfor 1 hour, fixed for 10 minutes in ice-cold methanol, washed in PBS,blocked in 10% BSA in PBS pH 7.2 before being incubated overnight withthe primary antibody (rat-anti-mouse-CD8α antibody, Sigma-Aldrich,Millipore).

The next morning the slides were washed in PBS and stained with asecondary antibody: goat-anti-rat-antibody-Alexa488 (Molecular Probe,Invitrogen) for 1 hour at room temperature. After another washing step,the slides were counterstained with 4′,6-diamidino-2-phenylindoledihydrochloride (DAPI) (Sigma-Aldrich, Millipore) and mounted inVectashield (Vector Laboratories). The slides were viewed and imagedusing a Zeiss Axioscope Microscope equipped with a mercury vapour lamp,appropriate filters and a ×20 apochromatic objective. Examples of imagesobtained from slides from the vehicle, MRx0518, and anti CTL4 animalsare shown (FIG. 1F—upper panels: DAPI staining, lower panels: CD8αstaining).

Fields of view were examined from 20 animals and imaged using manualexposure time. The number of animals and fields analysed are shown inthe following table:

Number of fields Number of Group analysed mice Vehicle 53 5 MRx0518 70 7anti 71 8 CTL4

The images were scored as follow: fields with ≤3 positive cells werescored as 0, whilst fields with more ≥3 cells were scored as 1. Theresults of this analysis are shown (FIG. 1G).

Ileum cryosections stained with anti-CD8α showed a higher number of CD8αpositive cells localized in the crypt region tissues from animalstreated with MRx0518 and anti-CTLA-4 compared to the vehicle group.

This observation is in line with CD8+ T cells being present in theintestine in case of infection or inflammatory microenvironment, as partof the immune response.

Antitumor Activity, LL/2 (LLC1) Model

The results are shown in FIG. 2 . Treatment with the bacterial strain ofthe invention led to a clear reduction in tumour volume relative to boththe negative controls.

Antitumor Activity, Hepal-6 Model

The results are shown in FIG. 3A. The untreated negative control doesnot appear as would be expected, because liver weight was lower in thisgroup than the other groups. However, the vehicle negative control andthe positive control groups both appear as would be expected, becausemice treated with vehicle alone had larger livers than mice treated withanti-CTLA4 antibodies, reflecting a greater tumour burden in the vehiclenegative control group. Treatment with the bacterial strain of theinvention led to a clear reduction in liver weight (and therefore tumourburden) relative to the mice in the vehicle negative control group.

Antitumor Activity, RENCA Model

The results are shown in FIG. 3B. Treatment with MRx0518 monotherapyreduced tumour volume with Test/Control of 51% (day 18) compared withthe vehicle-treated groups. Paclitaxel and anti-CTLA-4+anti-PDL-1 showedan (almost) complete reduction in tumour size at D18 and D22 compared toboth the untreated and vehicle groups.

These data indicate that strain MRX518 may be useful for treating orpreventing cancer, and in particular for reducing tumour volume inbreast, lung, kidney and liver cancers.

Example 2—PCR Gene Analysis

A pure culture of bacteria MRX518 was studied in a PCR gene analysis.There were two arms to the experiment: 1) MRX518 was co-cultured withhuman colonic cells (CaCo2) to investigate the effects of the bacteriaon the host, and 2) MRX518 was co-cultured on CaCo2 cells that werestimulated with IL1 to mimic the effect of the bacteria in aninflammatory environment. The effects in both scenarios were evaluatedthrough gene expression analysis. The results are shown below:

Fold Gene change Function CXCL3 28412.73 CXCR2 ligand, CXCL2 135.42CXCR2 ligand, 90% homology with CXCL1. CXCL9 34.76 CXCR3 ligand,primarily thought of as Th1 cell chemoattractant (inducible by IFN-g)IL8 31.81 Cytokine, chemoattractant (especially neutrophils), manyreceptors including CXCR1 and CXCR2/ CXCL1 16.48 CXCR2 ligand,stimulates cell proliferation as well as migration, overexpression isneuroprotective in EAE. CD40 14.33 Co-stimulatory molecule, route of Tcell dependent DC activation. TNF 13.50 Major proinflammatory cytokineIL17C 12.18 Promotes antibacterial response from epthielium, synergisticwith IL-22, CXCL10 10.66 Close homology with CXCL9, think also CXCR3ligand? HSPA1B 10.19 Heat shock protein NFKBIA 8.87 NFkB signalling;PI3K JUN 7.61 Antibacterial response; GPCR signalling. TNFAIP3 6.63 TNFsignalling DUSP1 6.36 Anti-inflammatory phosphatase, inactivates MAPKsJUNB 5.36 Transcription factor, JAK-STAT signalling BIRC3 4.86 Adherensjunctions, tight junctions DUSP2 4.59 Anti-inflammatory, inactivatesMAPK. IL32 4.29 Proinflammatory cytokine, induced by IFN-g, IL-18 DUSP53.12 Anti-inflammatory, inactivates MAPK FOS 3.03 Transcription factors,TLR signalling, forms part of AP-1 GADD45B 2.89 Cell growth andproliferation CLDN4 2.61 Tight junctions ADM 2.57 NFkB signalling KLF102.49 Cell arrest, TGF-b singllaing. DEFB4A −2.34 Antimicrobial peptideAPBA1 −2.53 Signalling IGFBP1 −2.72 Signalling pathway IL28B −2.73IFN-lambda, antiviral immune defence, IL10 −3.38 Anti-inflammatorycytokine NR4A1 −5.57 Nuclear receptor, anti-inflammatory, regulator of Tcell proliferation. T helper cell differentiation NOD2 −14.98 PRR,inflammasome activator, promotes autophagy INOS −26.88 Proinflammatory,generator of nitric oxide

These data appear to show two gene expression signatures—CXCR1/2 ligands(CXCL3, CXCL2, CXCL1, IL-8), which is associated with pro-inflammatorycell migration, and CXCR3 ligands (CXCL9, CXCL10), which is morespecifically indicative of IFN-γ-type responses, also supported byIL-32, which is IFN-γ-inducible.

Example 3—Stability Testing

A composition described herein containing at least one bacterial straindescribed herein is stored in a sealed container at 25° C. or 4° C. andthe container is placed in an atmosphere having 30%, 40%, 50%, 60%, 70%,75%, 80%, 90% or 95% relative humidity. After 1 month, 2 months, 3months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years, atleast 50%, 60%, 70%, 80% or 90% of the bacterial strain shall remain asmeasured in colony forming units determined by standard protocols.

Example 4—Cytokine Production in Immature Dendritic Cells Induced byMRX518 Compared to MRX518+LPS Summary

This study tested the effect of the bacterial strain MRX518 alone and incombination with lipopolysaccharide (LPS) on cytokine production inimmature dendritic cells.

A monocyte population was isolated from peripheral blood mononuclearcells (PBMCs). The monocyte cells were subsequently differentiated intoimmature dendritic cells. The immature dendritic cells were plated outat 200,000 cells/well and incubated with MRX518 at a final concentrationof 10⁷/ml, with the optional addition of LPS at a final concentration of100 ng/ml. The negative control involved incubating the cells with RPMImedia alone and positive controls incubated the cells with LPS at afinal concentration of 100 ng/ml. The cytokine content of the cells wasthen analysed.

Results

The results of these experiments can be seen in FIGS. 4 a-d . Theaddition of MRX518 alone leads to a substantial increase in the level ofcytokines IL-6 and TNF-α compared to the negative control (FIGS. 4 a andc ). The addition of LPS (positive control) leads to an increase in thelevel of IL-6 and TNF-α compared to the negative control but not IL-1β(FIG. 4 b ). A combination of MRX518 and LPS led to a synergisticincrease in the level of IL-1β produced (FIG. 4 d ).

Conclusion

MRX518 has the ability to induce higher IL-6 and TNF-α cytokineproduction in immature dendritic cells. The combination LPS and MRX518can increase the levels of cytokines IL-1α in immature dendritic cells.These data indicate that MRX518 alone or in combination with LPS canincrease inflammatory cytokines IL-1β, IL-6 and TNF-α, which promotesinflammation that can suppress cancer. Treatment with MRX518 alone or incombination with can induce cytokines that can limit tumour growth.

Example 5—Cytokine Production in THP-1 Cells Induced by MRX518 Comparedto MRX518+LPS Summary

This study tested the effect of bacterial strain MRX518 alone and incombination with LPS on cytokine production in THP-1 cells, a model cellline for monocytes and macrophages.

THF-1 cells were differentiated into MO medium for 48 h with 5 ng/mLphorbol-12-myristate-13-acetate (PMA). These cells were subsequentlyincubated with MRX518 at a final concentration of 10⁸/ml, with orwithout the addition of LPS at a final concentration of 100 ng/ml. Thebacteria were then washed off and the cells allowed to incubate undernormal growing conditions for 24 h. The cells were then spun down andthe resulting supernatant was analysed for cytokine content.

Results

The results of these experiments can be seen in FIGS. 5 a-c . Theaddition of MRX518 without LPS leads to an increase in the cytokinelevels of IL-1β, IL-6 and TNF-α compared to the no bacterial and thebacterial sediment controls. The addition of LPS and MRX518 leads to asynergistic increase in the production of cytokines.

Conclusion

MRX518 has the ability to induce cytokine production in THP-1 cells,which can be synergistically increased with the addition of LPS. Thesedata indicate that MRX518 alone or in combination with LPS can increaseinflammatory cytokines IL-1β, IL-6 and TNF-α, which promotesinflammation that can suppress cancer. Treatment with MRX518 alone or incombination with can induce cytokines that can limit tumour growth.

Example 6— Antitumour Activity of a Therapeutic Combination of MRX518and the PD-1 Inhibitor RMP1-14 or a CTLA-4 Inhibitor Summary

This study compared the anti-tumour activity of MRX518, the PD-1inhibitor RMP1-14, a CTLA-4 inhibitor and therapeutic combinations ofMRX518 with the PD-1 inhibitor RMP1-14 or the CTLA-4 inhibitor in micebearing EMT-6 tumour cells.

Materials

Test and reference substances—Bacterial strain #MRX518; Anti-PD-1antibody (clone: RMP1-14, catalog: BE0146, isotype: Rat IgG2a,Bioxcell); Anti-CTLA4 antibody (ref: BE0131, Bioxcell; clone: 9H10;reactivity: mouse; isotype: Hamster IgG1; storage conditions: +4° C.).

Test and reference substances vehicles—The MRX518 bacteria were grown ina bacterial culture medium (Yeast extract, Casitone, Fatty Acid medium(YCFA)) and kept as a glycerol stock at −80° C. The animals were dosedwith the bacteria according to the study protocol. The anti-PD1 andanti-CTLA-4 antibodies were diluted with PBS (ref: BE14-516F, Lonza,France) on each day of injection to mice.

Treatment doses—Bacteria: 2×10⁸ in 200 μL. The anti PD1-1 and anti CTLA4antibodies were administered at 10 mg/kg body weight according to themost recent body weight of mice.

Routes of administration—The bacterial composition was administered byoral gavage (per os, PO) via a gavage tube at a volume of 200 μL/inj.The anti PD-1 and anti CTLA-4 antibodies were injected into theperitoneal cavity of mice (Intraperitoneally, IP) at a volume of 10ml/kg adjusted to the most recent individual body weight of mice.

Cancer cell line and culture conditions—The cell line that was used inthis study is the EMT-6 cell line that was obtained from the ATCC(American Type Culture Collection, Manassas, Va., USA). The EMT-6 cellline was established from a transplantable murine mammary carcinoma thatarose in a BALB/cCRGL mouse after implantation of a hyperplastic mammaryalveolar nodule.

Tumor cells were grown as monolayer at 37° C. in a humidified atmosphere(5% CO₂, 95% air). The culture medium was RPMI 1640 containing 2 mML-glutamine (ref: BE12-702F, Lonza, Verviers, Belgium) supplemented with10% fetal bovine serum (ref: 3302, Lonza). EMT-6 tumor cells areadherent to plastic flasks. For experimental use, tumor cells weredetached from the culture flask by a 5-minute treatment withtrypsin-versene (ref: BE02-007E, Lonza), in Hanks' medium withoutcalcium or magnesium (ref: BE10-543F, Lonza) and neutralized by additionof complete culture medium. The cells were counted and their viabilitywas assessed by 0.25% trypan blue exclusion assay.

Use of animals—One hundred and thirty (130) healthy female Balb/C(BALB/cByJ) mice, 5-7 weeks old, were obtained from CHARLES RIVER(L'Arbresles) and maintained in SPF health status according to theFELASA guidelines. Animal housing and experimental procedures wererealized according to the French and European Regulations and NRC Guidefor the Care and Use of Laboratory Animals. Animals were maintained 3-4per cage in housing rooms under controlled environmental conditions:Temperature: 22±2° C., Humidity 55±10%, Photoperiod (12 h light/12 hdark), HEPA filtered air, 15 air exchanges per hour with norecirculation Animal enclosures were provided with sterile and adequatespace with bedding material, food and water, environmental and socialenrichment (group housing) as described: Top filter polycarbonateEurostandard Type III or IV cages, Corn cob bedding (ref: LAB COB 12,SERLAB, France), 25 kGy Irradiated diet (Ssniff® Soest, Germany),Complete food for immunocompetent rodents—R/M-H Extrudate, Sterile,filtrated at 0.2 μm water and Environmental enrichment (SIZZLE-drikraft—D20004 SERLAB, France). Animals are individually identified withRFID transponder and each cage was ladled with a specific code.Treatment of the animals started after one week of acclimation forbatches 2 and 3, or after three weeks of acclimation for batch 1.

Experimental Design and Treatments

On day −14 (D-14), 130 non-engrafted mice were randomized according totheir individual body weight into 3 groups of 30 animals and 4 groups of10 animals using Vivo Manager® software (Biosystemes, Couternon,France). The mice were separated into 3 batches of 10 animals pertreatment group (batch 1: 10 animals of groups 1, 2 and 3; batch 2: 10animals of groups 1, 2 and 3 and batch 3: 10 animals of groups 1 to 7)with different termination points from the start of the study: D-14 orD0.

At termination, batch 3 was split into 2 cohorts, due to termination andFACS analyses schedules; these were staggered over 1 day: D24/D25.Therefore, every cohort of animals had 5 animals per treatment group (4animals from cage one and one animal from cage 2). Based on the ethicalcriteria, if the tumor volume were higher than 1500 mm³, the selectionof the animals to be sacrifice on D24 and D25 is based on tumor volumeinstead of the cage. The experimental design is depicted in FIG. 7A andsummarized below:

-   -   1) Batch 1 (groups 1, 2 and 3) started treatment on D0 and was        culled at D14 (10 animals form groups 1 to 3). These did not        receive tumor cells and constituted the baseline group.    -   2) Batch 2 (group 1, 2 and 3) started treatment on D-14 and was        culled at D7 (10 animals form groups 1 to 3).    -   3) Batch 3 (groups 1 to 7) started treatment on D-14 and was        culled at D24/25 (10 animals form groups 1 to 7). The treatment        of anti PD-1 and Anti CTLA-4 started on D10.    -   On day 0 (D0) all mice of batches 2 and 3 (termination at day 7        and 24/25, respectively) were engrafted with EMT-6 tumour cells        by a subcutaneous injection of 1×10⁶ EMT-6 cells in 200 μL RPMI        1640 into the right flank (the 30 mice from batch 1, that were        sacrificed on D14, did not receive the tumour injection). The        mice were treated according to the following treatment schedule        groups (TWx2=twice a week):

Treatment Group No. Animals Treatment Dose Route Schedule 1 30 =Untreated — — — 10 batch 1 (+Tumour) 10 batch 2 10 batch 3 2 30 =Vehicle — PO Daily −14 to D0 10 batch 1 (YCFA) Daily −14 to D7 10 batch2 Daily −14 to D24/25 10 batch 3 3 30 = MRX518 (grown 2 × 10⁸ PO Daily−14 to D0 10 batch 1 from gly stock) Daily −14 to D7 10 batch 2 in YCFADaily −14 to D24/25 10 batch 3 4 10 batch 3 Anti-PD-1 + 10 mg/kg IP + POTWx2 from D10 YCFA YCFA Daily −14 to D24/25 5 10 batch 3 Anti-PD-1 + 10mg/kg + IP + PO TWx2 from D10 MRX518 2 × 10⁸ Bacteria Daily −14 bacteriato D24/25 6 10 batch 3 Anti-CTLA-4 + 10 mg/kg IP + PO TWx2 from D10 YCFAYCFA Daily −14 to D24/25 7 10 batch 3 Anti- CTLA-4 + 10 mg/kg + IP + POTWx2 from D10 MRX518 2 × 10⁸ Bacteria Daily −14 bacteria to D24/25

The following samples are collected throughout the experiment:

-   -   1. Feces (only for batch 3)—At three time points during the        study (D-15, D-1 and D22) faecal samples were collected from        eight identical mice per group (the equivalent of 80-100 mg or        6-7 pellets per mouse, but at least 3 faecal pellets), snap        frozen and stored at −80° C.    -   2. Blood—At the time of termination of the mice (D14 for batch1,        D7 for batch 2 and D25 for batch 3), approximately 1 mL of        intracardiac blood was collected from each animal into an EDTA        tube in terminal procedures under deep gas anesthesia. The blood        was centrifuged to obtain plasma, and the plasma stored at −80°        C.    -   3. Tumour and spleen—The tumour (on D and D24/D25) and the        spleens (on D7, D14 and D24/D25) from all mice were collected.        The tumour immune infiltrate cells in the tumour samples were        quantified by FACS analysis as described below.    -   4. Mesenteric lymph nodes—On D7, D14 and D24/D25 mesenteric        lymph nodes from all animals per groups and per time point were        collected and snap frozen at −80° C.    -   5. Intestine—At the time of euthanasia (D7, D14 and D24/D25),        several sections of the intestines from all mice per group and        per timing were collected and dissected. The caecal content was        harvested as well.

FACS Analysis

For analysis of tumor cells, tumors from all mice per groups and pertiming were collected at time of termination (on D7 and D24/25). All thetumors were collected in HBSS culture medium. The tumor immuneinfiltrate cells were quantified by FACS analysis from each collectedsample. Briefly, the collected samples were processed by mechanicdissociation and prepared in 100 μL staining buffer (PBS, 0.2% BSA,0.02% NaN₃). Then the antibodies directed against the chosen markerswere added, according to the procedure described by the supplier foreach antibody. All the antibodies except FoxP3 were for surface labelingand FoxP3 for intracellular labeling. The antibodies used for FACSanalysis are listed in the tables below:

Panel 1: panel T cells viability, CD45, CD3, CD4, CD8, CD25, FOXP3, PD1,B220 Reference Specificity and fluorochrome Isotype and specificityProvider 553052 CD4 PerCP mouse IgG2ak BD biosciences 553933 IgG2a PerCP— IgG2ak BD biosciences 562600 CD3 BV421 mouse IgG1k BD biosciences562601 IgG1 BV421 — IgG1k BD biosciences 130-110-665 CD45 Viogreen mouseREA737 Miltenyi Biotec 130-104-624 REA CTL universal VioGreen — REA293Miltenyi Biotec 563061 CD25 BV605 mouse IgG1, λ BD biosciences 562987IgG1 BV605 — IgG1λ BD biosciences 130-111-601 FoxP3** APC mouse REAMiltenyi Biotec 130-104-615 REA Control (I)** APC — REA/hIgG1 MiltenyiBiotec 564997 Fixable Viability Stain 700 eq AF700 — — BD biosciences130-109-250 CD8a APC-Vio770 mouse REA Miltenyi Biotec 130-104-634 REAAPC-Vio770 — REA Miltenyi Biotec 130-111-800 CD279 (=PD1) PE mouseREA802 Miltenyi Biotec 130-104-628 REA CTL universal PE — REA293Miltenyi Biotec 130-110-845 CD45R (B220) FITC mouse REA755 MiltenyiBiotec 130-104-626 REA CTL universal FITC — REA293 Miltenyi Biotec

Panel 2 tumor associated macrophages (TAM): viability, CD45, CD3, CD11b,Ly6C, F4/80, CD68, CD80, CD206, MHCII Reference Specificity andfluorochrome Isotype and specificity Provider 141704 CD206 FITC mouseIgG2a biolegend 553929 IgG2a FITC — IgG2ak BD biosciences 130-116-396CD80 PE mouse REA Miltenyi Biotec 130-104-628 REA CTL universal PE —REA/hIgG1 Miltenyi Biotec 130-109-289 CD11b PerCP-Vio700 mouse- REAMiltenyi Biotec human 130-104-620 REA Control (S) PerCP Vio700 — REAMiltenyi Biotec 130-116-530 CD3 PE-Vio770 mouse REA/hIgG1 MiltenyiBiotec 130-104-632 REA CTL universal PE-Vio770 — REA/hIgG1 MiltenyiBiotec 130-112-861 CD68* Vioblue mouse REA Miltenyi Biotec 130-104-625REA CTL universal* VioBlue — REA/hIgG1 Miltenyi Biotec 130-102-412 CD45Viogreen mouse IgG2b Miltenyi Biotec 130-102-659 IgG2b VioGreen — IgG2bMiltenyi Biotec 565694 Fixable Viability eq BV605 — — BD biosciencesStain 575V 130-102-379 F4/80/EMR1 APC mouse REA Miltenyi Biotec130-104-630 REA CTL universal APC — REA Miltenyi Biotec 130-112-233MHCII APC vio770 mouse REA/hIgG1 Miltenyi Biotec 130-104-634 REA CTLuniversal APC-Vio770 — REA/hIgG1 Miltenyi Biotec

The mixture was incubated for 20 to 30 minutes at room temperature inthe dark, washed, and re-suspended in 200 μL staining buffer. Allsamples were stored on ice and protected from light until FACS analysis.Tumor samples were also processed with control isotype antibodies. Thestained cells were analyzed with a CyFlow® space flow cytometer (LSR II,BD Biosciences) equipped with 3 excitation lasers at wavelengths 405,488 and 633 nm.

For analysis of intestine samples, the small intestine and the colon ofall mice per groups and per timing was collected at the time oftermination (on D7, D14 and D24/25). All the fresh tissues werecollected in HBSS culture medium. The immune cells in the lamina propriawere quantified by FACS analysis from each collected sample. The sampleswere processed as the tumor samples. The antibodies used for FACSanalysis are those of panel 1 listed above and those listed in the tablebelow (subsequent incubation of samples and analysis were as describedabove):

Panel 3: intestinal DCs: viability, CD45, CD3, CD11b, CD11c, MHC II,CD103 Reference Specificity and fluorochrome Isotype and specificityProvider 130-109-289 CD11b PerCP-Vio700 mouse- REA Miltenyi Biotec human130-104-620 REA Control (S) PerCP Vio700 — REA Miltenyi Biotec130-116-530 CD3 PE-Vio770 mouse REA/hIgG1 Miltenyi Biotec 130-104-632REA CTL universal PE-Vio770 — REA/hIgG1 Miltenyi Biotec 560583 CD11cAlexaFluor 700 mouse IgG1 BD bioscience 560555 IgG1 AlexaFluor 700 —IgG2 BD bioscience 130-102-412 CD45 Viogreen mouse IgG2b Miltenyi Biotec130-102-659 IgG2b VioGreen — IgG2b Miltenyi Biotec 565694 FixableViability eq BV605 — — BD biosciences Stain 575V 130-108-184 CD103 APCmouse REA Miltenyi Biotec 130-104-630 REA CTL universal APC — REA/hIgG1Miltenyi Biotec 130-112-233 MHCII APC vio770 mouse REA/hIgG1 MiltenyiBiotec 130-104-634 REA CTL universal APC-Vio770 — REA/hIgG1 MiltenyiBiotec 130-102-327 F4/80/EMR1 FITC mouse REA126 Miltenyi Biotec130-104-626 REA CTL universal FITC — REA293 Miltenyi Biotec

For analysis of spleen samples, the spleen of all mice per groups andper timing was collected at the time of termination (on D7, D14 andD24/25). All the spleens were collected in complete RPMI culture medium(10% dFBS, Penicillin/streptomycin 1%, 2 mM L-glutamine and 55 μM2-mercaptoethanol). The tumor immune infiltrate cells were quantified byFACS analysis from each collected sample after stimulation for 72 h withCD3 and CD28. Procedure: Splenocytes were cultured with either one oftwo stimulations (CD3/CD28, heat-killed MRx0518) and one negativecontrol. There was a ratio of 1:1 between the heat-killed MRx0518 andthe splenocytes per well. There was 1×10⁶ bacterial cells provided in 20μl of the heat-killed MRx0518 sample. The antibodies directed againstthe markers of panel 1 above were added to cell pellets from eachtreatment, according to the procedure described by the supplier for eachantibody. Subsequent incubation of samples and analysis were performedas described above.

Animal Monitoring

The viability and behaviour of the animals was recorded every day. Bodyweights were measured twice a week. The length and width of the tumourwas measured twice a week with callipers and the volume of the tumourwas estimated by the following formula:

${{Tumour}{volume}} = \frac{{width}^{2} \times {length}}{2}$

The treatment efficacy was assessed in terms of the effects of the testsubstance on the tumour volumes of treated animals relative to controlanimals. The following evaluation criteria of antitumor efficacy weredetermined using Vivo Manager® software (Biosystemes, Couternon,France):

-   -   1. Individual and/or mean (or median) tumour volumes. Mean        tumour volumes of groups 1 to 7 are depicted in FIG. 7B.    -   2. Tumour doubling time (DT).    -   3. Tumour growth inhibition (T/C %) defined as the ratio of the        median tumor volumes of treated versus control group, calculated        as follows (Dx=Day of measurement):

${T/C\%} = {\frac{{Median}{tumour}{volume}{of}{treated}{group}{at}D_{x}}{{Median}{tumour}{volume}{of}{vehicle}{treated}{group}{at}D_{x}} \times 100}$

-   -    The optimal value is the minimal T/C % ratio reflecting the        maximal tumour growth inhibition achieved. The effective        criteria for the T/C % ratio, according to NCI standards, is        42%.    -   4. Relative tumour volume (RTV) curves of test and control        groups, where the RTV is calculated as follows (Dx=Day of        measurement; DR=Day of randomization):

${RTV} = \frac{{TV}{at}D_{x}}{{TV}{at}D_{r}}$

-   -   5. Volume V and time to reach V are calculated. Volume V is        defined as a target volume deduced from experimental data and        chosen in exponential phase of tumour growth. For each tumour,        the closest tumour volume to the target volume V is selected in        tumour volume measurements. The value of this volume V and the        time for the tumour to reach this volume are recorded. For each        group, the mean of the tumour volumes V and the mean of the        times to reach this volume are calculated.

Example 7—CD8 Proliferation Assessment

To investigate the immunostimulatory effects of MRX518 and inhibitors ofthe invention, an in vitro assessment of the impact on CD8+ cellproliferation of MRX518 and the anti PD-1 checkpoint inhibitor MiltenyiBiotech CD279 in combination was conducted.

Peripheral blood mononuclear cells (PBMCs, cryopreserved from StemcellTechnologies, catalogue number: 70025), were removed from liquidnitrogen and allowed to rest overnight in a flask. A 96-well plate wascoated with CD3 antibody (ThermoFisher CD3 Monoclonal Antibody (OKT3),0.3 μs/ml) as one half of a mitogenic combination. Following the restingperiod, the PBMCs were counted and stained with fluorescent cell tracer(CellTrace™ Far Red Cell Proliferation Kit).

Ten sets of cells were prepared in this way. To nine of those sets, antiPD-1 antibody was added (from Miltenyi Biotech CD279 (PD1) purefunctional grade, 10 μs/ml). No anti PD-1 antibody was added to theadditional set, which served as a control set (referred to as Cell Set 1in the below table). All cell sets were then incubated for 1.5 hours.

Following the incubation period, bacterial test components were added toCell Sets 3 to 10 as shown in the following table:

Acronym as presented Cell Set Bacterial Component in FIG. 6 1 None, antiPD-1 free control CD3/CD28 2 None, anti PD-1 control anti-PD1 10 μg/ml(MY) 3 Heat Killed MRX518 at HKMRx0518 WT 1:1 a ratio of 1:1* 4 HeatKilled MRX518 at HKMRx0518 WT 10:1 a ratio of 10:1* 5 Heat Killed MRX518HKMRx0518 KO 1:1 with flagellin knockout** at a ratio of 1:1* 6 HeatKilled MRX518 HKMRx0518 KO 10:1 with flagellin knockout** at a ratio of10:1* 7 MRX518 supernatant at HK MRx0518 WT SN 1:1 a ratio of 1:1*** 8MRX518 supernatant at HK MRx0518 WT SN 10:1 a ratio of 10:1*** 9 MRX518flagellin HK MRx0518 KO SN 1:1 knockout supernatant** at a ratio of1:1*** 10 MRX518 flagellin HK MRx0518 KO SN 10:1 knockout supernatant**at a ratio of 10:1*** *Ratio of MRX518 cells:PBMC cells **A mutant ofMRX518 engineered to have a disrupted flagellar assembly was tested. Theflagellin is understood by the inventors to contribute to theimmunostimulatory effect of MRX518. ***For the 1:1 Multiplicity OfInfection (MOI), the supernatant was taken from the same number ofbacteria as the number of PBMCs treated with the supernatant. For theMOI of 10:1, the supernatant was taken from a highly concentratedbacterial culture, but the precise number of bacteria with respect tothe PBMCs was not measured.

Following the addition of the bacterial test components, a CD28 antibody(Thermofisher CD28 Monoclonal Antibody (CD28.2), 1 μg/ml) was added toeach of the cell sets as the other half of the mitogenic combination, totrigger proliferation. PDL-1 (R&D Systems, Recombinant Human PD-L1/B7-H1Fc Chimera, 10 μg/ml) was then added to each cell set.

The cell sets were then incubated for 5 days (37° C., 5% CO₂). Followingthe incubation, the cells were harvested and analysed by FACS accordingto cellular fluorescence imparted by the cell tracer, providing anindication of the number of cell divisions that had occurred in theincubation period. The results showing the percentages of cells groupedinto the number of divisions (from no cell division (NCD) to 4 celldivisions (4RCD)) are shown in FIG. 6 .

SEQUENCES

 (Enterococcus gallinarum 16S rRNA gene - AF039900) SEQ ID NO: 1   1 taatacatgc aagtcgaacg ctttttcttt caccggagct tgctccaccg aaagaaaaag  61 agtggcgaac gggtgagtaa cacgtgggta acctgcccat cagaagggga taacacttgg 121 aaacaggtgc taataccgta taacactatt ttccgcatgg aagaaagttg aaaggcgctt 181 ttgcgtcact gatggatgga cccgcggtgc attagctagt tggtgaggta acggctcacc 241 aaggccacga tgcatagccg acctgagagg gtgatcggcc acactgggac tgagacacgg 301 cccagactcc tacgggaggc agcagtaggg aatcttcggc aatggacgaa agtctgaccg 361 agcaacgccg cgtgagtgaa gaaggttttc ggatcgtaaa actctgttgt tagagaagaa 421 caaggatgag agtagaacgt tcatcccttg acggtatcta accagaaagc cacggctaac 481 tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tgtccggatt tattgggcgt 541 aaagcgagcg caggcggttt cttaagtctg atgtgaaagc ccccggctca accggggagg 601 gtcattggaa actgggagac ttgagtgcag aagaggagag tggaattcca tgtgtagcgg 661 tgaaatgcgt agatatatgg aggaacacca gtggcgaagg cggctctctg gtctgtaact 721 gacgctgagg ctcgaaagcg tggggagcga acaggattag ataccctggt agtccacgcc 781 gtaaacgatg agtgctaagt gttggagggt ttccgccctt cagtgctgca gcaaacgcat 841 taagcactcc gcctggggag tacgaccgca aggttgaaac tcaaaggaat tgacgggggc 901 ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc 961 ttgacatcct ttgaccactc tagagataga gcttcccctt cgggggcaaa gtgacaggtg1021 gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca1081 acccttattg ttagttgcca tcatttagtt gggcactcta gcgagactgc cggtgacaaa1141 ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg1201 tgctacaatg ggaagtacaa cgagttgcga agtcgcgagg ctaagctaat ctcttaaagc1261 ttctctcagt tcggattgta ggctgcaact cgcctacatg aagccggaat cgctagtaat1321 cgcggatcag cacgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac1381 cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt tttggagcca gccgcctaag1441 gtgggataga tgattggggt gaagtcgtaa caaggtagcc gtatcggaag gtgcggctgg1501  atcacc(consensus 16S rRNA sequence for Enterococcus gallinarum strain MRX518)SEQ ID NO: 2TGCTATACATGCAGTCGAACGCTTTTTCTTTCACCGGAGCTTGCTCCACCGAAAGAAAAAGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACACTATTTTCCGCATGGAAGAAAGTTGAAAGGCGCTTTTGCGTCACTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGATGAGAGTAGAACGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACTCTAGAGATAGAGCTTCCCCTTCGGGGGCAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCATCATTTAGTTGGGCACTCTAGCGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGAAGTACAACGAGTTGCGAAGTCGCGAGGCTAAGCTAATCTCTTAAAGCTTCTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTTGGAGCCAGCCGCCTAAGGTG

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1.-24. (canceled)
 25. A method of treating cancer in a subject in needthereof, comprising administering to said subject (i) a firstpharmaceutical composition that comprises a therapeutically effectiveamount of a bacteria strain of the genus Enterococcus, wherein saidbacteria strain comprises a 16S rRNA gene that has at least 95% sequenceidentity to the polynucleotide sequence of SEQ ID NO: 2, as determinedby a Smith-Waterman homology search algorithm using an affine gap searchwith a gap open penalty of 12 and a gap extension penalty of 2, and (ii)a second pharmaceutical composition that comprises a checkpointinhibitor, wherein said checkpoint inhibitor is an anti-ganglioside GD3antibody or a ganglioside GD3 binding fragment thereof, whether saidfirst pharmaceutical composition is administered to said subject by oraladministration and said second pharmaceutical composition isadministered to said subject by parenteral administration, and whereinsaid administering is effective to treat cancer in said subject.
 26. Themethod of claim 25, wherein said second pharmaceutical composition isadministered to said subject by subcutaneous or intravenousadministration.
 27. The method of claim 25, wherein said firstpharmaceutical composition is administered to said subject daily orevery other day.
 28. The method of claim 25, wherein said secondpharmaceutical composition is administered to said subject twice a week,once every 7 days, once every 14 days, once every 21 days, or once every28 days.
 29. The method of claim 25, wherein said first pharmaceuticalcomposition is administered to said subject daily and said secondpharmaceutical composition is administered to said subject every 14days.
 30. The method of claim 25, wherein said first pharmaceuticalcomposition is administered to said subject daily and said secondpharmaceutical composition is administered to said subject every 21days.
 31. The method of claim 25, wherein said first pharmaceuticalcomposition is administered to said subject daily and said secondpharmaceutical composition is administered to said subject every 28days.
 32. The method of claim 25, wherein said first pharmaceuticalcomposition is administered to said subject prior to firstadministration of said second pharmaceutical composition.
 33. The methodof claim 32, wherein said first pharmaceutical composition isadministered to said subject for at least 7, 14, 21, or 28 days prior tosaid first administration of said second pharmaceutical composition. 34.The method of claim 25, wherein said first pharmaceutical composition isadministered to said subject at least partially in parallel toadministration of said second pharmaceutical composition.
 35. The methodof claim 25, wherein said cancer is solid tumor cancer.
 36. The methodof claim 25, wherein said cancer is any one selected from the groupconsisting of lung cancer, breast cancer, kidney cancer, liver cancer,lymphoma, hepatoma, neuroendocrine cancer, melanoma, bladder cancer, andcolon cancer.
 37. The method of claim 36, wherein said cancer isnon-small cell lung cancer.
 38. The method of claim 25, wherein saidsubject is non-responsive or partially responsive to administering saidsecond pharmaceutical composition alone, as compared to saidadministering.
 39. The method of claim 38, wherein said subject has notbeen administered with an anti-cancer agent or therapy for treating saidcancer prior to said administering.
 40. The method of claim 25, whereinsaid subject is intolerant to a chemotherapy treatment.
 41. The methodof claim 25, wherein said administering is more effective to treatcancer as compared to administering said second pharmaceuticalcomposition alone or as compared to administering said firstpharmaceutical composition alone.
 42. The method of claim 25, whereinsaid bacterial strain is dried.
 43. The method of claim 25, wherein saidtherapeutically effective amount of said bacteria strain comprises fromabout 1×10³ to about 1×10¹¹ colony forming units (CFU)/g of saidbacteria strain with respect to the total weight of said firstpharmaceutical composition.
 44. The method of claim 25, wherein saidbacteria strain is capable of at least partially colonizing an intestineof said subject.